Rapid multiple panel of biomarkers in laboratory blood tests for TIA/stroke

ABSTRACT

A methods, kits and compositions for diagnosing a central nervous system disorder, particularly transient ischemic attack or stroke, comprising measuring the level of NR2A and/or NR2B NMDA receptor or fragment thereof, in a biological sample from a human subject, and optionally measuring other biomarkers such as homocysteine and glutamate. The method is particularly useful for identifying individuals that are at risk for stroke, and for diagnosing stroke in an emergency room setting.

RELATION TO PRIOR APPLICATIONS

[0001] This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Application No. 60/301,297, filed Jun. 27, 2001, and under35 U.S.C. §120 to U.S. Utility application Ser. No. 09/632,749, filedAug. 4, 2000 (currently pending), of which this application is acontinuation-in-part.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates generally to the diagnosis,management and therapy of central nervous system disorders such asstroke, transient ishemic attack, and traumatic brain injury. Inparticular, the invention relates to methods and kits for evaluatingthese central nervous system disorders, in order to better respond toepisodes of focal cerebral ishemia, and to best manage the riskassociated with future acute incidences.

[0004] 2. Background Information

[0005] Stroke or “brain attack” is clinically defined as a rapidlydeveloping syndrome of vascular origin that manifests itself in focalloss of cerebral function. In more severe situations, the loss ofcerebral function is global. A stroke occurs when the blood supply tothe part of the brain is suddenly interrupted (ischemic) or when a bloodvessel in the brain bursts, spilling blood into the spaces surroundingthe brain cells (hemorrhagic). The symptoms of stroke are easy to spot:sudden numbness or weakness, especially on one side of the body; suddenconfusion or trouble speaking or understanding speech; sudden troubleseeing in one or both eyes; sudden trouble walking; dizziness; or lossof balance or coordination. (National Institute of NeurologicalDisorders and Stroke, 2001). Stroke is the most common devastatingneurologic disease in the world, and the third leading cause of death inworld after heart disease and cancer. Despite recent progressunderstanding stroke mechanisms, stroke management is still not optimalfor a number of reasons.

[0006] The importance of promptly diagnosing a stroke after symptomsappear cannot be overstated. Delays in diagnosis and medicalintervention beyond 3 hours after stroke onset may contribute toclinical deterioration and disability. An early diagnosis enablesdoctors to more effectively choose the emergency intervention such asanti-platelet or/and neuroprotective therapy, and also to make betterprognoses of disease outcome. Successful treatment of stroke requiresrapid state diagnosis. The delay in achieving an accurate and certaindiagnosis wastes the limited amount of time available in which the braincan respond to reperfusion, and significantly increases the risk ofhemmorrhage after most of the permanent injury has occurred (Marler J.R. Annl. Emergency Med. 1999, 33: 450-451).

[0007] Unfortunately, however, many people who have a stroke either donot seek immediate medical care or suffer from delays in medical careeven in countries where stroke care is advanced, such as the UnitedStates and Europe (Alberts M J, Hademenos G, Latchaw R E, et al. JAMA2000; 23:3102-3109). Several clinical criteria can be employed todiagnose whether a patient is having a stroke, but even all thesecriteria do not always allow one to differentiate the episode from otherdisorders, such as epilepsy, syncope, and migraine (Toole J F.Cerebrovascular Disorders. 1999. Lippincott, Williams & Wilkins, NewYork, 5^(th) Ed., 542 p). Moreover, progressing stroke is only partiallypredictable based on clinical and neuroimaging data that is currentlyavailable to neurologists.

[0008] Transient ischemic attack (TIA) is a short-lived episode of focalneurologic deficit which often precedes the cerebral infarction of astroke. It occurs when the blood supply to part of the brain is brieflyinterrupted, and is typically accompanied by permanent brain damage(albeit less severe damage than normally results from a stroke). TIAsymptoms, which usually occur suddenly, are similar to those of strokebut do not last as long. Most symptoms of a TIA disappear within anhour, although they may persist for up to 24 hours. Symptoms caninclude: numbness or weakness in the face, arm, or leg, especially onone side of the body; confusion or difficulty in talking orunderstanding speech; trouble seeing in one or both eyes; and difficultywith walking, dizziness, or loss of balance and coordination. (NationalInstitute of Neurological Disorders and Stroke, 2001). Patients who havesuffered a TIA have 9.5 times greater risk of having a future strokethan those who have not had a TIA, and about one third of patients whosuffer a TIA will have an acute stroke in the future. (American StrokeAssociation, 2001). However, because the symptoms of TIA are short term,many patients do not recognize the event as a TIA or perceive the eventas a warning of a potentially impending stroke.

[0009] Standard treatments to reduce the risk of future stroke includethe use of antiplatelet agents, particularly aspirin. People with atrialfibrillation (irregular beating of the heart) may be prescribedanticoagulants. The most important treatable factors linked to TIAs andstroke are high blood pressure, cigarette smoking, heart disease,carotid artery disease, diabetes, and heavy use of alcohol. Lifestylechanges can often be implemented to reduce these factors. However, it isnecessary to diagnose the TIA as a warning sign of impending strokebefore such treatments can be administered. Therefore, a laboratoryblood test to detect TIA or stroke, or the risk of suffering a TIA orstroke in the future, would be of tremendous benefit.

[0010] During the past 5 years a number of molecular and immunochemicalassays have been evaluated for clinical use in neurology. (Schenone A.et.al. Current Opinion in Neurology. 1999, 12: 603-604; Honnorat J.J.Neurol. Neurosurg. Psychiatry. 1996, 61:270-278). At present, theThrombogene V and two Thrombx tests are available for diagnosingstroke/thrombosis from Athena Diagnostic. These tests evaluate thefrequent deep vein thrombosis and hypercoagulation states of patients toevaluate the need for intravenous anticoagulant therapy. The ThrombogeneV test detects the Factor V Leiden mutation by Polymerase Chain Reaction(PCR) in the blood of patients. The other two tests monitor changes ofdifferent blood coagulation markers: antithrombin III protein C, factorIX, and anticardiolipin antibodies (IgG, IgM, IgA) by use of ELISAtechnique. These tests thus reveal the hypercoagulation state as aresult of a thrombotic events, such as stroke stroke.

[0011] Stroke can be related to different types of venousthromboembolisms, which are common disorders with considerable morbidityand potential for mortality (Anderson, D.; Wells, P. Cur.Opinion inHemat. 2000, 7: 296-301). The biochemical marker: D-dimer, a breakdownproduct of a cross-linked fibrin blood clot that indicates theoccurrence of plasmin mediated lysis of cross-linked fibrin, has beenextensively evaluated for use in diagnostic tests for indicating acutevenous thromboembolism. Indeed, a fully automated, semi-quantative latexagglutination assays that uses turbimetric or agglutination endpointshas been developed that provides results within 20 minutes withsensitivity between 89% and 95% (Roussi J.; Bentolila L.; Contributionof D-dimer determination in the exclusion of deep venus thrombosis inspinal cord injury patients. Spinal Cord, 1999; v.37: p.548-552).Unfortunately, however, the presence of D-dimer may also be increased inother settings that result in fibrin generation, including recentsurgery, hemmorhage, trauma, cancer, and pregnancy (Anderson D R., WellsP S.; Thromb.Haemost.; 1999; 82:878-886).

[0012] However, the foregoing tests do not elucidate upon the TIA/strokemechanisms that are actually responsible for the damage associated withneurotoxic molecular events. It is necessary to find out specific andsensitive biomarkers which could be helpful to recognize initial braindamage and which could help to choose not only the appropriateanticoagulant treatment, but also emergency or regular neuroprotectivetherapy.

[0013] It is well known that two of the three leading causes of death,namely cardiovascular diaseses and stroke, are the end result ofatherosclerosis. Thus, it is not surprising that several biochemicalmarkers implicated in thromboembolic processes are also reported to beassociated with stroke and stroke risk. Among these are homocysteine,cholesterol and LDL (Cerebrovascular Disorders ed. by J. E. Toole.Lippincott Williams & Wilkins. 1999, pp.34-35), which are alsoclassified as risk factors to cardiovascular and cerebrovasculardiseases. (Hankey G J., and Eikelboom J W. Lancet. 354: 407-413 (1999).Approximately one fourth of patients with symptomatic atherosclerosishave elevated plasma homocysteine levels caused by various factors. Highlevels of homocysteine may run in families with increased susceptibilityto heart attack and stroke (Graham I. J.Ir.Call.Phys.Surg. 1995; 24:25-30). Elevated plasma homocysteine may be a causal and modifiablerisk-factor for ischemic stroke, but the results of previous studieshave been conflicting (Deulofeu V N R, Chamorro A, Piera C. Med Clin(Barc). 1998; 110: 605-608; Yamamoto T, Rossi S, Stiefel M F, DoppenbergE, Zauner A, Bullock R, Marmarou A. Acta Neurochir.Suppl.(Wien) 1999;75:17-19).

[0014] The neurotoxic effect of excitatory amino acids (glutamate,aspartate) in the brain has also been well documented. The results ofthis work show a correlation between glutamate content in the blood andthe severity of acute ischaemia (Castillo J, Dávalos A, Naveiro J, NoyaM. Stroke 1996, 27:1060-1065; Castillo J, Davalos A, Noya M. Lancet.1997; 349:79-83). Cerebral damage and its association with progressingstroke has been attributed to increased glutamate release, or lowglutamate reuptake, both in animals and in humans (Dávalos A, CastilloJ, Serena J, Noya M. Stroke 1997; 28:708-710).

[0015] However, only 56% of patients with progressing stroke arereported to have high glutamate content in their blood serum (DávalosA., Toni D., Iweins F., et al., 1999, 30: 2631-2636). Moreover, eventhough glutamate is considered the strongest biochemical predictor ofprogressing stroke (Davalos A, and Castillo J. In Book: CerebrovascularDisease. Current Med.Inc.: Philadelphia. 2000 Chapter 16, pp.169-181),this marker remains non-specific for TIA. Glutamate changes have alsobeen observed in the blood of patients with epilepsy and other nervoussystem disorders (Meldrum B S. J.Nutrition. 2000, 130:1007S-1015S).

[0016] Over the last three decades substantial progress has been made inelucidating the mechanisms by which cerebral ischemia leads to braindamage. The cellular and molecular mechanisms of cerebral ischemiaabnormalities have been better defined through the role of glutamate andglutamate receptors, one of the most distributed excitatoryneuroreceptors in brain, in regulating of initial stages of braindamage. Indeed, numerous molecular investigators consider glutamatereceptors to be one of the key biological receptors involved in themolecular mechanisms of TIA and stroke (Meldrum B S. J.Nutrition. 2000,130:1007S-1015S). According to a leading hypothesis, ischemia-inducedglutamate release activates these glutamate receptors. It has been shownthat glutamate and homocysteine (the sulfinic analog of aspartate)activate the glutamate binding site of NMDA receptors and participate inneurotoxic processes (Lipton S. A., Kim W. K., Choi Y. B., Kumar S., etal. PNAS. 1997, 94: 5923-5928).

[0017] Glutamate receptors are divided into two main groups: ionotropicand metabotropic. The ionotropic neuroreceptors are ligand-gated ionchannels that are subdivided into NMDA, AMPA and kainate receptorsubtypes. There are four NR2 subunits: NR2A, NR2B, NR2C and NR2D, whichis responsible for Ca²⁺-permeability regulation. NMDA receptors can bemodified by ischemia, resulting in changes of ion permeability and/orion selectivity.

[0018] Recent research findings indicate that the blood of patients withCNS disorders other than TIA or stroke exhibit properties ofautoimmunization to products of nerve cell degradation (Vincent A.,Oliver L., Pallace J. J Neuroimmun. 1999; 100: 169-180). For example, acorrelation between AMPA GluRl autoantibodies and common epilepsy hasbeen shown (Dambinova et al. J.Neurol. Sci.1997; 152: 93-97; Dambinovaet al. J. Neurochem. 1998;71: 2088-2093), as has a correlation betweenAMPA GluR 3 autoantibodies and Rasmussen's encephalitis (Rogers S W,Andrews P I, Gahring L C, et al. Science. 1995;265:648-651; Twyman R E,Gahring L C, Spiess J, Rogers S W. Neuron. 1995; 14:755-762; Gahring LC, Twyman R I, Greenlee J E, Rogers S W. Mol. Med. 1995; 1:245-253).

[0019] In a similar vein, several researchers have reported an increasein NMDA receptor synthesis, the appearance of high levels of receptorantigen, and the generation of autoantibodies to the receptors duringthe initial stages of cerebral ishemia (Gusev et al., J.Neurol.&Psych.1996, 5:68-72; Dambinova et al. J.Neurol.Sci. 1997, 152:93-97;Dambinova et al. J.Neurochem. 1998, 71: 2088-2093). Acting on thisresearch, one company developed a laboratory kit (cerebral ischaemia(CIS)-test) that detects autoantibodies to the N-terminus domain of theNR2A subunit in the blood of patients with TIA or stroke (Gusev E. I.,Skvortsova V I, Alekseev A A, Izykenova G A, Dambinova S A. S. SKorsakov's J.Neurol.& Psych. 1996; 5:68-72). The N-terminus domain ofthe NR2A subunit of NMDA receptors was selected as the immunoreactiveepitope on the basis of molecular biological and experimental studiesshowing that this epitope is the most immunoreactive region of thereceptor (Dambinova S A, Izykenova G A. J.High Nervous Activity. 1997;47:439-446).

[0020] More recently, researchers have reported a correlation betweenthe effectiveness of a stroke treatment regimen and the levels ofautoantibodies to the NR2A and NR2B subunits of NMDA. In particular,these researchers have reported increased titers of autoantibodies tothe NR2A and NR2B subunits of NMDA in the blood of patients severelyaffected by stroke, and a reduction of the autoantibodies, accompaniedby an improvement in neurological function, during therapy by glycine—anon-specific agonist of NMDA receptors (Gusev et.al. CerebrovascularDiseases. 2000, 10: 49-60). Patients that responded positively toglycine had lower autoantibody titers than patients who were nottreated, and had levels of autoantibodies that were close to the levelsof autoantibodies in control subjects.

[0021] Unfortunately, the use of NR2A and NR2B autoantibodies in thediagnosis of stroke or TIA does not provide a real-time assessment ofthe damage being done by a stroke or TIA. Rather, because of the timethe immune system requires to mount an immune response, and to generateNR2A and NR2B autoantibodies, methods that test for these antibodies atbest provide a delayed assessment of the extent and severity of strokeor TIA.

[0022] Investigators from Canada (Hill M. D., Jackowski G., Bayer N.,Lawrence M., Jaeschke R. Can.Med.Assoc.J. 2000, 163: 1139-1140) haveproposed a new diagnostic laboratory assay for differentiating strokesubtype. They designed a preliminary prospective cohort study to test apanel of biochemical markers (neuron-specific enolase[NSE], myelin basicprotein [MBP], S-100 [betta] protein and thrombomodulin [Tm]) in bloodsamples from patients with acute ischemic stroke. These markers werechosen because they cover important cellular components of the brainthat might be damaged in acute stroke. The 4 biochemical markers wereassayed using a standard ELISA technique.

[0023] The results of this investigation demonstrated elevated levels ofNSE in 89% of the patients admitted in hospitals, Tm in 43%, MBP in 39%and S-100 [beta] in 32%. At least one of the markers was elevated onadmission in 93% of the acute stroke patients. By stroke type, 100% ofthe patients with lacunar stroke, 100% of those with posteriorcirculation stroke and 90% of those with partial anterior circulationstroke had elevated NSE levels on admission. Conversely, none of thepatients with lacunar stroke had en elevated S-100[beta] level initiallyor subsequently. Peak levels of NSE, S-100 [beta] and MBP, but not ofTm, were significantly correlated with admission NIHSS scores (p<0.05).

[0024] For stroke, 3 hours is an outside limit for administeringappropriate therapies. The focus must change from extensive evaluationbefore any action to a well-planned acute emergency therapy developedusing an appropriate diagnostic strategy. Every future advance toimprove the outcome after TIA/stroke will depend on a fast initialresponse-within minutes and not hours (Marler J. R. Annl. Emergency Med.1999, 33: 450-451). Therefore, it is especially important to develop afast and simple method (within one hour) of detecting brain and bloodbiomarkers capable of recognizing the initial processes of TIA/strokebefore irreperable ischemic damage ensues.

OBJECTS OF INVENTION

[0025] Therefore, it is an object of the invention to providebiochemical methods and kits for diagnosing central nervous systemdisorders such as TIA and stroke.

[0026] It is another object of the present invention to improve upon theaccuracy of currently available methods for diagnosing TIA and stroke,and to more accurately diagnose TIA and stroke to the exclusion of othernervous system disorders or traumatic brain injury.

[0027] It is still another object of the present invention to providemethods of diagnosing stroke using biochemical markers that distinguishbetween hemorrhagic and ischemic stroke.

[0028] Still another object of the invention is to provide biochemicalanalyses of the extent and progression of TIA or stroke, or theinfarction resulting from the TIA or stroke.

[0029] It is another object of the present invention to provide rapidbiochemical methods and kits for diagnosing TIA and stroke, to providereal-time assessments of TIA or stroke, within a window of time thatpermits effective therapeutic intervention.

[0030] It is another object of the present invention to provide rapidand inexpensive biochemical methods and kits for diagnosing TIA andstroke, which can be performed at frequent intervals to monitor theprogression of a TIA or stroke, or the effectiveness of therapyadministered against TIA or stroke.

[0031] Still another object of the present invention is to providediagnostic methods and kits for assessing the risk of incurring a TIA orstroke, and for monitoring the remission of risk factors for TIA orstroke.

[0032] Still another object of the invention is to provide a panel ofrapid multiple panel of biomarkers for assessing the nature, severityand progression of TIA or stroke, and thereby to enable a more effectiveselection of intervention therapy.

SUMMARY OF THE INVENTION

[0033] It has unexpectedly been discovered that levels of circulatingNMDA receptor proteins or fragments thereof can be assessed usingdiagnostic kits and processes, and that levels of these proteins orfragments can be used to clinically evaluate patients suffering fromishemic central nervous system disorders such as stroke or TIA. Whenanalyzed in combination with other biomarkers for stroke and TIA, suchas the thromboembolic marker homocysteine, or the excititory amino acidglutamate, these proteins can diagnose the existence of a stroke withremarkable accuracy (generally greater than 89%). In contrast, theefficacy of single parameters for early diagnosis of stroke is 58% forglutamate, 66% for homocysteine, and 79% for NMDA receptors. The rapidevaluation of these neural ischemic biomarkers in an emergency roomsetting will greatly enhance the confidence of physicians whendiagnosing stroke or TIA, and significantly improve the speed at whichtherapy against the stroke or TIA can be administered.

[0034] The biomarkers also yield extensive evidence about the nature ofthe stroke or TIA and the type therapy which should be administered. Forexample, the respective levels of biomarkers can be evaluated todetermine whether the patient is suffering an ishemic or hemmorhagicstroke, or whether the patient is suffering from a traumatic braininjury. The data from the biomarkers can also be used to monitor orevaluate the progression of the ishemic episode, as well as the damagethat has resulted as a consequence of the ischemia. High levels of allparameters reflect the neurological deficit and may be also used forprognosis of disease outcome. Moreover, a relationship has been observedbetween the respective levels of the biomarkers and the degree ofthromboembolic and neurotoxicity in brain processes under the stroke.Once again, these relationships can be put to extensive use whenevaluating the choice of emergency therapy in short time frames, such asanti-platelet and neuroprotective therapy. The data can be usedindependently of other diagnostic strategies, but preferably forms anintegral part of a comprshensive diagnostic strategy employingconventional diagnostic techniques.

[0035] The data obtained from the NMDA biomarkers, especially whencombined with data from other biomarkers such as glutamate andhomocysteine, can also be used to monitor the efficacy of a treatmentregime. It has surprisingly been found that the NMDA biomarkers providereal time evidence of neurotoxicity, and that reductions in levels ofcirculating NR1A or NR2A NMDA receptors or fragments thereof correspondwell with reductions in neurotoxic mechanisms. By obtaining data atappropriate intervals using rapid laboratory techniques such as latexagglutination, one is able to monitor the progression of the episode inresponse to the therapeutic regime.

[0036] A latex agglutination technique has also been developed whichdramatically increases the speed of diagnosis obtained by the methods ofthis invention, and thereby improves the effectiveness of the methods inemergency-room settings. The technique can be adapted for use in thedetection of NMDA receptors, homocysteine, glutamate, or any othersuitable biomarker against central nervous system disorders. Using thelatex agglutination technique, one is able to provide real-timebiochemical diagnosis and monitoring of TIA/stroke patients (withinabout 30 minutes), and thereby dramatically improve the effectiveness ofresponse to TIA/stroke. This is surprising because these biomarkers arenaturally occurring and, in contrast to viruses for which latexagglutination methods were originally developed, show much lowerstrengths of association with their corresponding antibodies.

[0037] This semi-quantitative method gives reliable data quickly in aformat that is simple for interpretation. Surprisingly, the techniqueshows greater accuracy than even well established methods based uponHPLC and ELISA. The application of the latex agglutination technique tothe analysis of brain biomarkers for stroke will decrease the cost ofanalysis, provide the opportunity to monitor real-time progress of atreatment procedure, and allow physicians to determine the efficacy ofmedication administered in the treatment of TIA or stroke.

[0038] The methods of the present invention also can be employed in anon-emergency setting, when evaluating the risk that an individual willsuffer a stroke or TIA. In addition, based upon results showing anincreased risk of suffering TIA or stroke, prevention therapy can beadministered, and the effectiveness of the therapy monitored using themethods of the present invention.

[0039] The invention also relates to indirect methods for measuringlevels of NR2A and NR2B NMDA receptor proteins or fragments thereof.Thus, analytical techniques can be used to evaluate indirect measures ofNR2A and NR2B NMDA receptor proteins or fragments thereof, such asautoantibodies specific for the proteins, or cDNA that encodes for theproteins.

[0040] Additional advantages of the invention will be set forth in partin the description which follows, and in part will be obvious from thedescription, or may be learned by practice of the invention. Theadvantages of the invention will be realized and attained by means ofthe elements and combinations particularly pointed out in the appendedclaims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention, as claimed.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The present invention may be understood more readily by referenceto the following detailed description of preferred embodiments of theinvention and the Examples included therein. Before the present methodsand techniques are disclosed and described, it is to be understood thatthis invention is not limited to specific analytical or syntheticmethods as such may, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting.

[0042] Definitions and use of Terms

[0043] As used in this specification and in the claims which follow, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “afragment” includes mixtures of fragments, reference to “an cDNAoligonucleotide” includes more than one oligonucleotide, and the like.

[0044] An analogue of a protein, peptide, or polypeptide means aprotein, peptide, or polypeptide that contains one or more amino acidsubstitutions, deletions, additions, or rearrangements. For example, itis well known in the art of protein biochemistry that an amino acidbelonging to a grouping of amino acids having a particular size orcharacteristic (such as charge, hydrophobicity, and hydrophilicity) canoften be substituted for another amino acid without altering theactivity of the protein, particularly in regions of the protein that arenot directly associated with biological activity. Thus, an analogue ofan NMDA receptor or fragment thereof is useful in the present inventionif it includes amino acid substitutions, deletions, additions orrearrangements at sites such that antibodies raised against the analogueare still specific against the NMDA receptor or fragment.

[0045] Preferably, an NMDA analogue has at least 80%, 85%, 90%, or 95%amino acid identity with naturally occurring NMDA. Amino acid identityis defined by a analoguey comparison between the analogue and naturallyoccurring NMDA. The two amino acid sequences are aligned in such a waythat maximizes the number of amino acids in common along the length oftheir sequences; gaps in either or both sequences are permitted inmaking the alignment in order to maximize the number of common aminoacids. The percentage amino acid identity is the higher of the followingtwo numbers: (1) the number of amino acids that the two polypeptideshave in common with the alignment, divided by the number of amino acidsin the NMDA analogue or fragment thereof, multiplied by 100, or (2) thenumber of amino acids that the two polypeptides have in common with thealignment, divided by the number of amino acids in naturally occurringNMDA or fragment thereof, multiplied by 100.

[0046] NMDA derivatives, and derivatives of NMDA fragments, includenaturally occurring NMDA and NMDA analogues and fragments thereof thatare chemically or enzymatically derivatized at one or more constituentamino acids, including side chain modifications, backbone modifications,and N- and C-terminal modifications, by for example acetylation,hydroxylation, methylation, amidation, phosphorylation or glycosylation.The term also includes NMDA salts such as zinc NMDA and ammonium NMDA.

[0047] A protein or peptide is measured “directly” in the sense that theprotein or peptide is itself measured in the biological sample, asopposed to some other indirect measure of the protein or peptide such asautoantibodies to the protein or peptide, or cDNA associated with theexpression of the protein or peptide.

[0048] The term “antibody” is intended to be synonymous with“immunoglobulin.” As used herein, the term “antibody” is meant toinclude both the native antibody, and biologically active derivatives ofantibodies, such as, for example, Fab′, F(ab′)₂ or Fv as well assingle-domain and single-chain antibodies. A biologically activederivative of an antibody retains the ability to bind antigen.

[0049] General Discussion

[0050] The present disclosure describes diagnostic and therapeuticapplications that result from the realization that genetic or accidentalincrease of NMDA receptors synthesis in the brain reflects aneurological ischemic deficit, and may be used for early diagnoses ofstroke or TIA. NMDA receptors that are abnormally expressed in the brainare quickly metabolized and, following penetration of the blood brainbarrier, these metabolic destruction products enter the circulatorysystem. The immune system recognizes these peptides and proteinfragments as foreign antigens and responds by generating autoantibodiesto them.

[0051] In one aspect of the present invention, the correlation betweenincreased NMDA receptor synthesis, and the appearance of high levels ofthe receptors in blood sera of individuals during the initial stages ofcerebral ischemia, is used for diagnostic and therapeutic applications.Experiments in rats with focal ischemia have demonstrated that NR2A mRNAexpression in the cortex and hippocampus can be measured within twohours of the onset of the ischemic episode, and thus provide anopportunity for real time measurement of ischemic processes and damageresulting therefrom. At the same time, meaningful expression of NR2C andNR2D mRNA is not observed in brain structures that showed no changes inNR1 mRNA expression in rat ischemic brain. These changes in NR2-receptormRNA expression in the early stages of ischemia are observed prior tomorphological evidence of neuronal damage or appearance ofautoantibodies to them in blood serum specimens.

[0052] Thus, in one aspect the present invention provides a method fordiagnosing a central nervous system disorder comprising measuring thelevel of NR2A and/or NR2B NMDA receptor or fragment thereof in abiological sample. Elevated levels of NR2A and NR2B NMDA receptors arespecific to brain injury, and are expressed in ischemic brain tissue athigher rates than other NMDA receptors, and thus are uniquely suited forassessing ischemic brain episodes such as TIA or stroke. Baseline levelsfor determining whether the measured levels are elevated, and henceindicative of a central nervous system disorder, can be obtained frompopulation norms or, preferably, from a patient's own test history.

[0053] The biological sample tested for the receptor or fragment can bederived from blood, urine, blood plasma, blood serum, cerebrospinalfluid, saliva, perspiration, or brain tissue. In a preferred embodiment,the biological sample is a blood sample. In an even more preferredembodiment the biological sample is a blood sample diluted to a ratio offrom about 1:2 to about 1:32 (v:v).

[0054] Immunoassay techniques are generally preferred for measuring theproteins or peptides of the present invention, as discussed in greaterdetail herein, although other analytical techniques are also availableas known to those skilled in the art, such as HPLC. The amino acidsequences of the NR2A and NR2B subunits, and antigenic fragmentsthereof, are recited in SEQ ID NOS.1, 2, 3, 10, 11, and 12, and anyfragment of these sequences can be employed in methods for directlydetecting the receptors as long as sufficient antigenicity ismaintained. However, when using immunoassays it has been found that theantigenic determinants are concentrated in the N-terminal domain of theNR2A and/or NR2B NMDA receptor, and that antibodies raised against theN-terminal domains and fragments thereof should be employed for optimaltest results. The inventors have sequenced the amino acid chain of theN-terminal domains for these receptors, and set forth the sequences asSEQ ID NOS. 2 and 11, respectively, at the end of this document.

[0055] In a preferred embodiment, other biomarkers of central nervoussystem disorders are also measured to improve the accuracy of thediagnosis, and to provide further information about the nature,severity, or progression of the disorder. Particularly useful markersare directly implicated in the NMDA receptor pathway, and includenaturally occurring agonists and antagonists of the NMDA receptors. Anexemplary antagonist is glycine. Exemplary agonists include glutamate,polyglutamate, aspartate, polyaspartate, homocysteine, andpolyhomocysteine. A particularly preferred agonist for measuring theactivity of the receptors is glutamate or polyglutamate.

[0056] In another embodiment, thromboembolic biomarkers are measured toobtain a simultaneous reading of the likelihood for clotting in thebrain. Exemplary thromboembolic biomarkers include homocysteine orpolyhomocysteine.

[0057] Titers of higher than 2.63 for combined levels of NR2A and NR2B,especially when combined with titers higher than 3.34 for glutamateand/or 2.23 for homocysteine, are remarkably predictive of theoccurrence of stroke and typically justify immediate therapeuticintervention for the TIA or stroke or risk of stroke. These titers canbe translated into absolute concentrations by reference to the exampleshereof.

[0058] The methods of the present invention are preferably performed bydirectly measuring the levels of NR2A and/or NR2B biomarkers in aselected biological sample, using immunoassay techniques employingantibodies raised against the biomarkers, or through quantitativetechniques such as HPLC. However, it is also possible to measure thepresence of the NR2A and/or NR2B biomarkers indirectly. This can be doneby directly measuring autoantibodies of the biomarkers, or by directlymeasuring the cDNA nucleic acid intermediates involved in expression ofthese biomarkers. If autoantibodies are measured, they are preferablymeasured using one or more antigenic fragments of the NR2A and/or NR2Breceptors as the target of the antibody, as opposed to a whole NR2Aand/or NR2B protein. Healthy persons generally have NR2A autoantibodiesin an amount of about 1.0-2.0 ng/ml. Healthy persons generally have NR2AcDNA levels of about 1.0-1.5 pg/ml.

[0059] Latex Agglutination and Other Diagnostic Techniques

[0060] A number of immunoassays can be employed in accordance with theprinciples of the present invention. Examples include radioimmunoassays,enzyme immunoassays (e.g. ELISA), immunofluorescence,immunoprecipitation, latex agglutination, hemagglutination, andhistochemical tests. A particularly preferred method, however, becauseof its speed and ease of use, is latex agglutination.

[0061] Latex agglutination assays have been described in Beltz, G. A. etal., in Molecular Probes: Techniques and Medical Applications, A.Albertini et al., eds., Raven Press, New York, 1989, incorporated hereinby reference. In the latex agglutination assay, antibody raised againsta particular biomarker is immobilized on latex particles. A drop of thelatex particles is added to an appropriate dilution of the serum to betested and mixed by gentle rocking of the card. With samples lackingsufficient levels of the biomarkers, the latex particles remain insuspension and retain a smooth, milky appearance. However, if biomarkersreactive with the antibody are present, the latex particles clump intovisibly detectable aggregates.

[0062] An agglutination assay can also be used to detect biomarkerswherein the corresponding antibody is immobilized on a suitable particleother than latex beads, for example, on gelatin, red blood cells, nylon,liposomes, gold particles, etc. The presence of antibodies in the assaycauses agglutination, similar to that of a precipitation reaction, whichcan then be detected by such techniques as nephelometry, turbidity,infrared spectrometry, visual inspection, colorimetry, and the like.

[0063] The term latex agglutination is employed generically herein torefer to any method based upon the formation of detectableagglutination, and is not limited to the use of latex as theimmunosorbent substrate. While preferred substrates for theagglutination are latex based, such as polystyrene and polypropylene,particularly polystyrene, other well-known substrates include beadsformed from glass, paper, dextran, and nylon. The immobilized antibodiesmay be covalently, ionically, or physically bound to the solid-phaseimmunoadsorbent, by techniques such as covalent bonding via an amide orester linkage, ionic attraction, or by adsorption. Those skilled in theart will know many other suitable carriers for binding antibodies, orwill be able to ascertain such, using routine experimentation.

[0064] Thus, in one embodiment, the method of measuring the NR2A and/orNR2B NMDA receptor, fragment therof, or other biomarker is by latexagglutination comprising:

[0065] (i) contacting the biological sample with poly- or monoclonalantibodies bound on an agglutinating carrier to target biomakers for asufficient time period and under conditions to promote agglutination;and

[0066] (ii) reading a signal generated from the agglutination; whereinthe amount of signal detected correlates to the titer of biomarkerspresent in the sample.

[0067] The reaction is preferably read macroscopically against a darkbackground for a sufficient time period. The method preferably yields aclinically useful reading within about 30 minutes or less.

[0068] It has been experimentally found that latex beads having a meandiameter of from about 0.25 to about 0.4 μm are particularly preferredin the practice of this invention. The poly- or monoclonal antibodiesare preferably present in a ratio with the latex beads of about 1:1.

[0069] Latex beads having the foregoing characteristics can be preparedgenerally by adding antibodies to the target biomarker to a carriersolution that contains a 1% concentration (by weight) of latex beads,until the concentration of the antibodioes in the carrier solutionreaches about 2 mg/ml, and allowing the ingredients a sufficient time tocovalently link, typically about 1 hour, in the presence of a linkingagent such as glutaraldehyde.

[0070] Conventional methods can be used to prepare the antibodies. Forexample, by using a peptide of a NMDA protein, polyclonal antisera ormonoclonal antibodies can be made using standard methods. A mammal,(e.g., a mouse, hamster, or rabbit) can be immunized with an immunogenicform of the peptide (preferably the NR2A and/or NR2B receptor, anantigenic determinant of the NR2A and/or NR2B receptor, or an analogueor derivative thereof) which elicits an antibody response in the mammal.Techniques for conferring immunogenicity on a peptide includeconjugation to carriers or other techniques well known in the art. Forexample, the peptide can be administered in the presence of adjuvant.The progress of immunization can be monitored by detection of antibodytiters in plasma or serum. Standard ELISA or other immunoassayprocedures can be used with the immunogen as antigen to assess thelevels of antibodies. Following immunization, antisera can beadministered and, if desired, polyclonal antibodies isolated from thesera.

[0071] To produce monoclonal antibodies, antibody producing cells(lymphocytes) can be harvested from an immunized animal and fused withmyeloma cells by standard somatic cell fusion procedures thusimmortalizing these cells and yielding hybridoma cells. Such techniquesare well known in the art, (e.g., the hybridoma technique originallydeveloped by Kohler and Milstein (Nature 256, 495-497 (1975)) as well asother techniques such as the human B-cell hybridoma technique (Kozbor etal., Immunol. Today 4, 72 (1983)), the EBV-hybridoma technique toproduce human monoclonal antibodies (Cole et al. Monoclonal Antibodiesin Cancer Therapy (1985) Allen R. Bliss, Inc., pages 77-96), andscreening of combinatorial antibody libraries (Huse et al., Science 246,1275 (1989)]. Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with the peptide and themonoclonal antibodies can be isolated. Therefore, the invention alsocontemplates hybridoma cells secreting monoclonal antibodies withspecificity for NR2A or NR2B NMDA proteins or fragments thereof asdescribed herein.

[0072] When the NR2A and/or NR2B receptors are detected indirectly, bymeasuring the cDNA expression of the NR2A and/or NR2B receptors, themeasuring step in the present invention may be carried out bytraditional PCR assays such as cDNA hybridization, Northern blots, orSouthern blots. These methods can be carried out using oligonucleotidesencoding the polypeptide antigens of the invention. Therefore, in oneembodiment the methods are performed employing oligonucleotides thatencode the amino acid sequence of SEQ ID NO: 2, which is preferablyrepresented by nucleotides 371-1978 of SEQ ID NO: 6. More preferably,the nucleic acid construct comprises a oligonucleotide consisting of anucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3,which is preferably represented by oligonucleotides 1790-1852 of SEQ IDNO: 7.

[0073] Thus, in one embodiment the methods of this invention includemeasuring an increase of NR2A and/or NR2B cDNA expression by contactingthe total DNA isolated from a biological sample with oligonucleotideprimers attached to a solid phase, for a sufficient time period. Inanother preferred embodiment, NR2A and/or NR2B cDNA expression ismeasured by contacting an array of total DNA bound to a solid matrixwith a ready-to-use reagent mixture containing oligonucleotide primersfor a sufficient time period. Expressed NR2A cDNA is revealed by thecomplexation of the cDNA with an indicator reagent that comprises acounterpart oligonucleotide to the cDNA attached to a signal-generatingcompound. The signal-generating compound is preferably selected from thegroup consisting of horseradish peroxidase, alkaline phosphatase,urinase and non-enzyme reagents. The signal-generating compound is mostpreferably a non-enzyme reagent.

[0074] In a preferred embodiment, the solid phase is a polymer matrix.More preferably, the polymer matrix is polyacrylate, polystyrene, orpolypropylene. In one preferred embodiment the solid phase is amicroplate. In another preferred embodiment, the solid phase is anitrocellulose membrane or a charged nylon membrane.

[0075] As mentioned above, the methods of performing the presentinvention also may be performed by measuring the levels ofautoantibodies specific for the NR2A and/or NR2B subunits. Theseautoantibodies may be measured by any suitable immunoassay such as, forexample, a radioimmunoassay, an immunofluorescence assay, anenzyme-linked immunosorbent assay (ELISA), an immunocytochemical assay,and immunoblotting. In a preferred embodiment, the antigen to which theanti-NR2A and/or NR2B autoantibodies bind is a polypeptide or proteinfragment of the N-terminal domain of the NR2A and/or NR2B receptor. Morepreferably, the antigen comprises a polypeptide or protein fragment ofamino acid SEQ ID NO:2, 3, 4, 11, 12, or 13, or a suitable analogue orderivative thereof.

[0076] Thus, in yet another embodiment the methods of the presentinvention are performed by measuring the levels of anti-NR2A and/oranti-NR2B autoantibodies, by contacting a biological sample with apolypeptide or protein fragment of the NR2A and/or NR2B receptor(preferably the N-terminal domain) (or an analogue or derivativethereof) attached to a solid phase, for a sufficient time period andunder conditions to allow a complex to form between any NR2A and/or NR2Bautoantibodies which may be present in the sample and the polypeptide orprotein fragment, contacting the complex with an indicator reagentcomprising a secondary antibody specific for the species of the mammalattached to a signal-generating compound (or for the polypeptide orprotein fragment); and measuring the signal generated. The peptide canbe obtained directly from biological samples, by recombinant DNAtechniques, or by direct chemical synthesis. The signal-generatingcompound is preferably selected from horseradish peroxidase, alkalinephosphatase, and urinase. More preferably, the signal-generatingcompound is horseradish peroxidase. Most preferably, the indicatorreagent is rabbit anti-human IgG attached to horseradish peroxidase. Theamount of signal detected is correlated to the amount of anti-NR2Aand/or NR2B autoantibodies present in the biological sample.

[0077] In this method it is preferred that the solid phase be a polymermatrix. More preferably, the polymer matrix is selected from the groupconsisting of polyacrylate, polystyrene, and polypropylene. In onepreferred embodiment the solid phase is a microplate. In anotherpreferred embodiment, the solid phase is a nitrocellulose membrane or acharged nylon membrane.

[0078] The immunosorbent of the present invention for measuring levelsof autoantibody can be produced as follows. A fragment of the receptorprotein is fixed, preferably by covalent bond or an ionic bond, on asuitable carrier such as polystyrene or nitrocellulose. If the standardpolystyrene plate for immunological examinations is employed, it is frstsubjected to the nitration procedure, whereby free nitrogroups areformed on the plate surface, which are reduced to amino groups andactivated with glutaric dialdehyde serving as a linker. Next thethus-activated plate is incubated with about 2 to 50 nM of the targetpeptide for the purpose of chemically fixing the respective immunogenicfragment of the receptor protein for a time and at a temperaturesufficient to assure fixation (i.e. for about 16 hours at 4° C.).

[0079] The amount of protein below 2 nM affects adversely thereliability of the findings, whereas its amount exceeding 50 nM isinexpedient due to an increase in the nonspecific binding ofautoantibodies with the immunosorbents. The plate is then washed with anaqueous solution of sodium boron hydride and an aqueous solution ofsodium chloride, vacuum-dried, enclosed in a hermetically sealedpackage, and put under storage at 4° C.

[0080] It is also practicable to produce the immunosorbent by fixing therespective fragment of the receptor protein on nitrocellulose strips byvirtue of ionic interaction. The respective fragment of the receptorprotein isolated from the mammals' brain is applied to nitrocelluloseand incubated for 15 min at 37° C. Then nitrocellulose is washed with a0.5% solution of Tween-20, and the resultant immunosobent is dried atroom temperature and stored in dry place for one year period.

[0081] Emergency Room Diagnosis and Prognosis

[0082] As mentioned above, the methods of the present invention areespecially well suited for use in emergency room settings. There are tworeasons for this. First, the method is extremely useful in an emergencyroom setting because NR2A and NR2B NMDA receptor levels are elevated ata very early stage of ishemic insult, and thus provide a real timeindication of neurotoxic events. This is in contrast to autoantibodieswhich require that an immune response first be mounted by the insultedorganism.

[0083] The second reason the method is useful in an emergency roomsetting is the speed and ease with which the latex agglutinationprocedure can be employed. Using the latex agglutination processesdescribed herein, one is able to turn laboratory results around often inless than 30 or even 20 minutes. Thus, using the methods of the presentinvention real-time data can be obtained that permits a therapeuticresponse within the window for an effective response to stroke.

[0084] Therefore, in one embodiment the invention provides a method fordiagnosing the existence of a central nervous system disorder such asTIA or stroke, further comprising withdrawing the biological sample froma human subject, wherein the biological sample is withdrawn within threehours of the onset of symptoms of the central nervous system disorder.In still another embodiment of the invention, the amount of time elapsedbetween withdrawing the biological sample from the subject, anddetecting or measuring the presence or quantity of the NR2A and/or NR2BNMDA receptor, is less than about one hour, 45 minutes, or 30 minutes.

[0085] One of the principal adavntages of the present invention is theability to distinguish ischemic episodes such as stroke from other braininjuries such as traumatic brain injury. Thus, in another embodiment,the invention provides a method for diagnosing the existence of TIA orstroke further comprising evaluating from the level of NR2A and/or NR2BNMDA receptor whether the brain injury is a traumatic brain injury orstroke/TIA, and administering traimatic brain injuty or stroke/TIAtherapy as appropriate.

[0086] Another advantage of the methods of the present invention whichis extremely useful in an emergency room setting, is the ability todetermine from the test data the type of stroke involved. In particular,if a stroke is suspected, the method will help diagnose whether thestroke is an ischemic or hemorrhagic insult. Thus, in another embodimentthe invention provides a method for diagnosing the existence of TIA orstroke further comprising, when the diagnosis confirms a stroke,evaluating from the level of NR2A and/or NR2B NMDA receptor whether thestroke is ischemic or hemmorhagic and administering ischemic orhemmorhagic stroke therapy as appropriate.

[0087] Another advantage of the present invention is the ability toevaluate infarction volume and extent of neurotoxicity from NMDAexpression data. NMDA receptor expression research in an animal model ofmiddle carotid artery occlusion has been employed to demonstrate suchcorrelation. Thus, in still another embodiment the invention provides amethod for diagnosing the existence of TIA or stroke further comprising,if TIA and/or stroke is confirmed, evaluating from the level of NR2Aand/or NR2B NMDA receptor cranial infarct volume, and administeringtherapy appropriate to the infarct volume.

[0088] Moreover, one can periodically repeat the procedure, to providecontinuous monitoring of a patient's state as interventional therapy isadministered, to monitor the effectiveness of a particular therapeuticregime. In this embodiment, it is preferable for the mammal to beconcurrently undergoing treatment for the disorder. More preferably, thesamples are collected at intervals from about 20 min to about 1 month.Even more preferably, the interval is from about 20 min. to about 2hours. Most preferably the samples are collected at an interval of about30 minutes. Thus, in still another embodiment the invention provides amethod for diagnosing the progression of TIA or stroke furthercomprising detecting or measuring the presence or quantity of a NR2Aand/or NR2B NMDA receptor in a biological sample one or more additionaltimes, at a frequency of less than about 6 hours.

[0089] Primary Care Physician Setting

[0090] In another application the method is used in a clinical settingto determine an individual's risk of stroke, or to monitor theeffectiveness of risk reduction therapies. As mentioned above, a numberof therapies can be employed to reduce the risk of stroke in anindividual. The use of antiplatelet agents, particularly aspirin, is astandard treatment for patients at risk for stroke. People with atrialfibrillation (irregular beating of the heart) may be prescribedanticoagulants. The most important treatable factors linked to TIAs andstroke are high blood pressure, cigarette smoking, heart disease,carotid artery disease, diabetes, and heavy use of alcohol. Medical helpis available to reduce and eliminate these factors. Lifestyle changessuch as eating a balanced diet, maintaining healthy weight, exercising,and enrolling in smoking and alcohol cessation programs can also reducethese factors. When these therapies are administered it is desirable todetermine the effectiveness of the therapy.

[0091] Therefore, in one embodiment the invention provides a method forevaluating an individual's risk for TIA or stroke comprising measuringlevels of NR2A and/or NR2B NMDA receptors or fragments thereof in abiological sample from the individual, and comparing the levels to abaseline level. In one embodiment the baseline levels are derived frompopulation averages. In another embodiment the baseline levels arederived from the individual's own medical history.

[0092] In another embodiment the method is performed more than once tomonitor the reduction or increase in risk for stroke or TIA, optionallyin conjunction with the administration of risk reduction therapy. In oneembodiment the method is performed at a frequency of from about one weekto about six months. In another embodiment the method is performed at afrequency of from about one month to about three months.

[0093] In a particularly preferred embodiment other biomarkers are alsomeasured to assess the risk for stroke or TIA. Particularly preferredbiomarkers for risk of stroke or TIA are glutamate and homocysteine.

[0094] Novel Kits of the Present Invention

[0095] In another embodiment the invention provides kits for diagnosingcentral nervous system disorders such as TIA, stroke, and traumaticbrain injury. NR2A and/or NR2B antibodies or antigens may beincorporated into immunoassay diagnostic kits depending upon whetherautoantibodies or NMDA receptors are being measured. A first containermay include a composition comprising an antigen or antibody preparation.Both antibody and antigen preparations should preferably be provided ina suitable titrated form, with antigen concentrations and/or antibodytiters given for easy reference in quantitative applications.

[0096] The kits may also include an immunodetection reagent or label forthe detection of specific immunoreaction between the provided antigenand/or antibody, as the case may be, and the diagnostic sample. Suitabledetection reagents are well known in the art as exemplified byradioactive, enzymatic or otherwise chromogenic ligands, which aretypically employed in association with the antigen and/or antibody, orin association with a second antibody having specificity for firstantibody. Thus, the reaction is detected or quantified by means ofdetecting or quantifying the label. Immunodetection reagents andprocesses suitable for application in connection with the novel methodsof the present invention are generally well known in the art.

[0097] The reagents may also include ancillary agents such as bufferingagents and protein stabilizing agents, e.g., polysaccharides and thelike. The diagnostic kit may further include where necessary agents forreducing background interference in a test, agents for increasingsignal, apparatus for conducting a test, calibration curves and charts,standardization curves and charts, and the like.

[0098] In a more particular aspect the invention relates to a rapidmultiple panel containing antibodies to the thromboembolic andneurotoxicity biomarkers glutamate, homocysteine and NMDA receptors thatemploys latex agglutination. Thus, in one embodiment the inventionprovides a kit for diagnosing central nervous system disorderscomprising: (1) an agglutinating immunosorbent for NR2A and/or NR2B NMDAreceptors or fragments thereof, and (2) a control such as saline or aknown concentration of NR2A and/or NR2B receptor or fragment thereof. Ina more preferred embodiment the kit further comprises an agglutinatingimmunosorbent for another biomarker for TIA/stroke, such as an agonistor antagonist of NR2A and/or NR2B, a thromboembolic marker, or moreparticularly glutamate or polyglutamate, and/or an agglutinatinghomocysteine or polyhomocysteine. The agglutinating immunosorbent ispreferably of the type discussed in greater detail above.

[0099] In another embodiment the invention relates to a kit fordetecting NR2A and/or NR2B receptors or fragments thereof that does notemploy latex agglutination. Thus, in another embodiment the inventionprovides a kit for diagnosing central nervous system disorderscomprising: (1) an immunosorbent for NR2A and/or NR2B NMDA receptors orfragments thereof, and (2) an indicator reagent comprising secondaryantibodies attached to a signal generating compound. The secondaryantibodies can be specific for the receptor or fragrnent, or for theprimary antibodies in the immunosorbent. In a preferred embodiment thekits further comprise an immunosorbent for glutamate or polyglutamate,and/or an immunosorbent for homocysteine or polyhomocysteine, andsecondary antibodies against the glutamate and/or homocysteine, or tothe primary antibodies on the immunosorbents against the glutamate orhomocysteine. The immunosorbent preferably comprises anti-antibodies forthe biomarkers bound to a solid support.

[0100] In another aspect the present invention relates to a test-kitthat relies upon PCR amplification for measuring NR2A and/or NR2Blevels. Thus, in another embodiment the invention provides a kitcomprising: (a) one or more oligonucleotide primers (preferably of SEQID NO: 8) attached to a solid phase, (b) indicator reagent attached to asignal-generating compound capable of generating a detectable signalfrom oligonucleotides, and (c) a control sample (i.e. template cDNA).The reagents may also include ancillary agents such as buffering agents,polymerase agents, and the like. The diagnostic kit may further include,where necessary, other members of the signal-producing system of whichsystem the detectable group is a member (e.g., enzyme and non-enzymesubstrates), agents for reducing background interference in a test,agents for increasing the signal, apparatus for conducting a test, andthe like.

[0101] In another embodiment of test-kit comprises (a) a solid phase towhich biological fluids for receiving total DNA including NR2A cDNAcould be attached, (b) oligonucleotide primers, preferably in aready-to-use PCR buffer, and (c) a control sample (i.e. template cDNA).Ancillary agents as described above may similarly be included.

[0102] In another embodiment the invention provides a diagnostic kit fordetecting NR2A and/or NR2B autoantibodies comprising (a) a polypeptideof the N-terminal domain of the NR2A and/or NR2B receptor, fragmentthereof, or analog or derivative thereof, (b) an indicator reagentcomprising a secondary antibody specific for the autoantibody or thepolypeptide attached to a signal-generating compound; and (c) a controlsample, such as a known concentration of NR2A and/or NR2B polyclonalantibodies. The reagents may also include ancillary agents such asbuffering agents and protein stabilizing agents, e.g., polysaccharidesand the like. The diagnostic kit may further include, where necessary,other members of the signal-producing system of which system thedetectable group is a member (e.g., enzyme and non-enzyme substrates),agents for reducing background interference in a test, agents toincrease the signal, apparatus for conducting a test, calibration andstandardization information or instructions, and the like.

[0103] Novel Compositions of the Invention

[0104] The methods of the present invention rely upon a series of novelcompositions which themselves form a part of the invention. Thus, in oneseries of embodiments the invention provides an isolated polypeptidefragment of the NR2A and/or NR2B NMDA receptor, comprising:

[0105] 1. An antigenic determinant of the NR2A NMDA receptor,

[0106] 2. An antigenic determinant of the NR2B NMDA receptor,

[0107] 3. The N-terminal domain of the NR2A NMDA receptor,

[0108] 4. The N-terminal domain of the NR2B NMDA receptor,

[0109] 5. SEQ ID NO. 2,

[0110] 6. SEQ ID NO. 3,

[0111] 7. SEQ ID NO. 4,

[0112] 8. SEQ ID NO. 11,

[0113] 9. SEQ ID NO. 12, and

[0114] 10. SEQ ID NO. 13,

[0115] or an antigenic fragment, analog, or derivative thereof. Inanother series of embodiments the invention provides any of theforegoing polypeptides linked covalently to a distinct antigenicdeterminant, such as human serum albumin. In still another series ofembodiments the invention provides any of the foregoing polypeptideslinked to any of the immunosorbent materials discussed above. Theimmunosorbent can be in the form of a bead for latex agglutination, inthe size ranges discussed above, or in the form of a synthetic plate forconventional immunoassay analysis. The polypeptide can be linked to theimmunosorbent using any conventional means of linkage, includingcovalent linkage, ionic linkage, and adsorption.

[0116] In another series of embodiments the present invention relates tothe novel monoclonal and polyclonal antibodies specific for and/orraised against the foregoing polypeptides, including the foregoingpolypeptides linked to distinct antigenic determinants. Thus, in oneembodiment the invention provides non-human antibodies against any ofthe foregoing peptides or polypeptides or antigenic fragment, analog, orderivative thereof. In another embodiment the invention providesimmunosorbents to which such antibodies are linked.

[0117] In another series of embodiments the present invention providesoligonucleotides that encode the foregoing peptides and polypeptides andfragments, analogs, and derivatives thereof, and to recombinantexpression vectors that include such oligonucleotides. Sucholigonucleotides include, without limitation, the oligonucleotidesdefined by SEQ ID NO:6, 7, 14, and 15, and fragments thereof whichencode antigenic determinants.

[0118] In still another embodiment the present invention relates toisolated oligonucleotide sequences that are useful in the cDNA PCRanalytical techniques of the present invention. Thus, the inventionfurther provides oligonucleotides comprising the nucleotide sequences ofSEQ ID NOS:7, 8, 9, 15, 16, and 17.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

[0119] The features, aspects, and advantages of the present inventionwill become better understood with regard to the following sequencelistings where, in the sequence the recited amino acid positionnumbering reflects that used throughout this document.

[0120] SEQ ID NO: 1. shows the full-length amino acid sequence of themature NR2A receptor subunit, as follows:

[0121] SEQUENCE LISTING

[0122] PEPTIDE

[0123] Homo sapiens glutamate receptor. ionotropic, N-methyl D-aspartate2A

[0124] Science 256:1217-1221(1992)

[0125] NCBI/NM 000833.2 1``````````11`````````21`````````31 1 MGRLGYWTLLVLPALLVWRD PAQNAAAEKG PPALNIAVLL 41`````````51 GHSHDVTERE LRNLWGPEQA 6061 TGLPLDVNVV ALLMNRTDPK SLITHVCDLM SGARIHGLVF GDDTDQEAVA QMLDFISSQT 120121 FIPILGIHGG ASMJMADKDP TSTFFQFGAS IQQQATVMLK IMQDYDWHVF SLVTTIFPGY180 181 RDFISFIKTT VDNSFVGWDM QNVITLDTSF EDAKTQVQLK KJHSSVILLYCSKDEAVLJL 240 241 SEARSLGLTG YDFFWIVPSL VSGNTELIPK EFPSGLISVSYDDWDYSLEA RVRDGLGILT 300 301 TAASSMLEKF SYIPEAKASC YGQAEKPETPLHTLHQFMVN VTWDGKDLSF TEEGYQVHPR 360 361 LVVIVLNKDR EWEKVGKWENQTLSLRHAVW PRYKSFSDCE PDDNHLSIVT LEEAPFVIVE 420 421 DIDPLTETCVRNTVPCRKFV KINNSTNEGM NVKKCCKGFC IDILKKLSRT VKFTYDLYLV 480 481TNGKHGKKVN NVWNGMIGEV VYQRAVMAVG SLTINEERSE VVDFSVPFVE TGISVMVSRS 540541 NGTVSPSAFL EPFSASVWVM MFVMLLIVSA IAVFVFEYFS PVGYNRNLAK GKAPHGPSFT600 601 IGKAIWLLWG LVFNNSVPVQ NPKGTTSKIM VSVWAFFAVI FLASYTANLAAFMIQEEFVD 660 661 QVTGLSDKKF QRPHDYSPPF RFGTVPNGST ERNIRNNYPYMHQYMTRFNQ RGVEDALVSL 720 721 KTGKLDAFIY DAAVLNYKAG RDEGCKLVTIGSGYIFASTG YGIALQKGSP WKRQIDLALL 780 781 QFVGDGEMEE LETLWLTGICHNEKNEVMSS QLDIDNMAGV FYMLAAAMAL SLITFIWEHL 840 841 FYWKLRFCFTGVCSDRPGLL FSISRGIYSC IHGVHIEEKK KSPDFNLTGS QSNMLKLLRS 900 901AKNISNMSNM NSSRMDSPKR ATDFIQRGSL IVDMVSDKGN LIYSDNRSFQ GKDSIFGDNM 960961 NELQTFVANR HKDNLSNYVF QGQHPLTLNE SNPNTVEVAV STESKGNSRP RQLWKKSMES1020 1021 LRQDSLNQNP VSQRDEKTAE NRTHSLKSPR YLPEEVAHSD ISETSSRATCHREPDNNKNH 1080 1081 KTKDNFKRSM ASKYPKDCSD VDRTYMKTKA SSPRDKIYTIDGEKEPSFHL DPPQFVENIT 1140 1141 LPENVGFPDT YQDHNENFRK GDSTLPMNRNPLHNEDGLPN NDQYKLYAKH FTLKDKGSPH 1200 1201 SEGSDRYRQN STHCRSCLSNLPTYSGHFTM RSPFKCDACL RMGNLYDIDE DQMLQETGNP 1260 1261 ATREEVYQQDWSQNNALQFQ KNKLRINRQH SYDNILDKPR EIDLSRPSRS ISLKDRERLL 1320 1321EGNLYGSLFS VPSSKLLGNK SSLFPQGLED SKRSKSLLPD HASDNPFLHT YGDDQRLVIG 13801381 RCPSDPYKHS LPSQAVNDSY LRSSLRSTAS YCSRDSRGHS DVYISEHVMP YAANKNTMYS1440 1441 TPRVLNSCSN RRVYKKMPSI ESDV

[0126] SEQ ID NO:2. shows the amino acid sequence of the auto-antigenicregion of the N-terminal domain of the NR2A subunit, as follows:

[0127] SEQ ID NO:2

[0128] HOMO SAPIENS PAQNAAAEKG PPALNIAVLL GHSHDVTERE LRNLWGPEQA                      60 61 TGLPLDVNVV ALLMNRTDPK SLITHVCDLM SGARIHGLVFGDDTDQEAVA QMLDFISSQT 120 121 FIPILGIHGG ASMIMADKDP TSTFFQFGASIQQQATVMLK IMQDYDWHVF SLVTTIFPGY 180 181 RDFISFIKTT VDNSFVGWDMQNVITLDTSF EDAKTQVQLK KIHSSVILLY CSKDEAVLIL 240 241 SEARSLGLTGYDFFWIVPSL VSGNTELIPK EFPSGLISVS YDDWDYSLEA RVRDGLGILT 300 301TAASSMLEKF SYIPEAKASC YGQAEKPETP LHTLHQFMVN VTWDGKDLSF TEEGYQVHPR 360361 LVVIVLNKDR EWEKVGKWEN QTLSLRHAVW PRYKSFSDCE PDDNHLSIVT LEEAPFVIVE420 421 DIDPLTETCV RNTVPCRKFV KINNSTNEGM NVKKCCKGFC IDILKKLSRTVKFTYDLYLV 480 481 TNGKHGKKVN NVWNGMIGEV VYQRAVMAVG SLTINEERSEVVDFSVPFVE TGISVMVSRS 540 541 NGTVSPSAFL EPESAS

[0129] SEQ ID NO:3; shows a 21 amino acid antigenic peptide,corresponding to a fragment of the NR2A N-terminal domain.another suchpeptide (21 amino acids derived from the NR2A sequence and an N-terminalCys for attachment to a carrier protein), as follows:

[0130] SEQ ID NO:3

[0131] Homo sapiens

[0132] NGMIGEVVYQRAVMAVGSLTI

[0133] SEQ ID NO:4. shows a 22 amino acid antigenic peptide,corresponding to a fragment of the NR2A N-terminal domain.another suchpeptide, modified by an N-terminal Cys for attachment to a carrierprotein):

[0134] Artificial Sequence

[0135] CNGMIGEVVYQRAVMAVGSLTI

[0136] FULL

[0137] BASE COUNT ORIGIN

[0138] Homo sapiens glutamate receptor, ionotropic, N-methyl D-aspartate2A (GRIN 2A) mRNA

[0139] SEQ ID NO:5. shows the Oligonucleotide position numbering usedthroughout in reference to NR2A oligonucleotide sequences, as follows:

[0140] SEQ ID NO:5

[0141] Science 256:1217-1221(1992)

[0142] May 22, 1992

[0143] NIGB/NM_(—)000833 1 atcatgggac cgggtgagcg ctgagaatcg cggccgcagccatcagccct ggagatgacc 61 aggageggec actgctgaga actatgtgga gagaggctgcgagccctgct gcagagcctc 121 cggctgggat agccgccccc cgtgggggcg atgcggacagcgcgggacag ccaggggagc 181 gcgctggggc cgcagcatgc gggaacccgc taaacccggtggctgctgag gcggccgaga 241 tgctcgtgcg cgcagcgcgc cccactgcat cctcgaccttctcgggctac agggaccgtc 301 agtggcgact atgggcagag tgggctattg gaccctgctggtgctgccgg cccttctggt 361 ctggcgcggt ccggcgccga gcgcggcggc ggagaagggtccccccgcgc taaatattgc 421 ggtgatgctg ggtcacagcc acgacgtgac agagcgcgaacttcgaacac tgtggggccc 481 cgagcaggcg gcggggctgc ccctggacgt gaacgtggtagctctgctga tgaaccgcac 541 cgaccccaag agcctcatca cgcacgtgtg cgacctcatgtccggggcac gcatccacgg 601 cctcgtgttt ggggacgaca eggaccagga ggccgtagcccagatgctgg attttatctc 661 ctcccacacc ttcgtcccca tcttgggcat tcatgggggcgcatctatga tcatggctga 721 caaggatccg acgtctacct tcttccagtt tggagcgtccatccagcagc aagccacggt 781 catgctgaag atcatgcagg attatgactg gcatgtcttctccctggtga ccactatctt 841 ccctggctac agggaattca tcagcttcgt caagaccacagtggacaaca gctttgtggg 901 ctgggacatg cagaatgtga tcacactgga cacttcctttgaggatgcaa agacacaagt 961 ccagctgaag aagatccact cttctgtcat cttgctctactgttccaaag acgaggctgt 1021 tctcattctg agtgaggccc gctcccttgg cctcaccgggtatgatttct tctggattgt 1081 ccccagcttg gtctctggga acacggagct catcccaaaagagtttccat cgggactcat 1141 ttctgtctcc tacgatgact gggactacag cctggaggcgagagtgaggg acggcattgg 1201 catcctaacc accgctgcat cttctatgct ggagaagttctcctacatcc ccgaggccaa 1261 ggccagctgc tacgggcaga tggagaggcc agaggtcccgatgcacacct tgcacccatt 1321 tatggtcaat gttacatggg atggcaaaga cttatccttcactgaggaag gctaccaggt 1381 gcaccccagg ctggtggtga ttgtgctgaa caaagaccgggaatgggaaa aggtgggcaa 1441 gtgggagaac catacgctga gcctgaggca cgccgtgtggcccaggtaca agtccttctc 1501 cgactgtgag ccggatgaca accatctcag catcgtcaccctggaggagg ccccattcgt 1561 catcgtggaa gacatagaco ccctgaccga gacgtgtgtgaggaacaccg tgccatgtcg 1621 gaagttcgtc aaaatcaaca attcaaccaa tgaggggatgaatgtgaaga aatgctgcaa 1681 ggggttctgc attgatattc tgaagaagct ttccagaactgtgaagttta cttacgacct 1741 ctatctggtg accaatggga agcatggcaa gaaagttaacaatgtgtgga atggaatgat 1801 cggtgaagtg gtctatcaac gggcagtcat ggcagttggctcgctcacca tcaatgagga 1861 acgttctgaa gtggtggact tctctgtgcc ctttgtggaaacgggaatca gtgtcatggt 1921 ttcaagaagt aatggcaccg tctcaccttc tgcttttctagaaccattca gcgcctctgt 1981 ctgggtgatg atgtttgtga tgctgctcat tgtttctgccatagctgttt ttgtctttga 2041 atacttcagc cctgttggat acaacagaaa cttagccaaagggaaagcac cccatgggcc 2101 ttcttttaca attggaaaag ctatatggct tctttggggcctggtgttca ataactccgt 2161 gcctgtccag aatcctaaag ggaccaccag caagatcatggtatctgtat gggccttctt 2221 cgctgtcata ttcctggcta gctacacagc caatctggctgccttcatga tccaagagga 2281 atttgtggac caagtgaccg gcctcagtga caaaaagtttcagagacctc atgactattc 2341 cccacctttt cgatttggga cagtgcctaa tggaagcacggagagaaaca ttcggaataa 2401 ctatccctac atgcatcagt acatgaccaa atttaatcagaaaggagtag aggacgcctt 2461 ggtcagcctg aaaacgggga agctggacgc tttcatctacgatgccgcag tcttgaatta 2521 caaggctggg agggatgaag gctgcaagct ggtgaccatcgggagtgggt acatctttgc 2581 caccaccggt tatggaattg cccttcagaa aggctctccttggaagaggc agatcgacct 2641 ggccttgctt cagtttgtgg gtgatggtga gatggaggagctggagaccc tgtggctcac 2701 tgggatctgc cacaacgaga agaacgaggt gatgagcagccagctggaca ttgacaacat 2761 ggcgggcgta ttctacatgc tggctgccgc catggcccttagcctcatca ccttcatctg 2821 ggagcacctc ttctactgga agctgcgctt ctgtttcacgggcgtgtgct ccgaccggcc 2881 tgggttgctc ttctccatca gcaggggcat ctacagctgcattcatggag tgcacattga 2941 agaaaagaag aagtctccag acttcaatct gacgggatcccagagcaaca tgttaaaact 3001 cctccggtca gccaaaaaca tttccagcat gtccaacatgaactcctcaa gaatggactc 3061 acccaaaaga gctgctgact tcatccaaag aggttccctcatcatggaca tggtttcaga 3121 taaggggaat ttgatgtact cagacaacag gtcctttcaggggaaagaga gcatttttgg 3181 agacaacatg aacgaactcc aaacatttgt ggccaaccggcagaaggata acctcaataa 3241 ctatgtattc cagggacaac atcctcttac tctcaatgagtccaacccta acacggtgga 3301 ggtggccgtg agcacagaat ccaaagcgaa ctctagaccccggcagctgt ggaagaaatc 3361 cgtggattcc atacgccagg attcactatc ccagaatccagtctcccaga gggatgaggc 3421 aacagcagag aataggaccc actccctaaa gagccctaggtatcttccag aagagatggc 3481 ccactctgac atttcagaaa cgtcaaatcg ggccacgtgccacagggaac ctgacaacag 3541 taagaaccac aaaaccaagg acaactttaa aaggtcagtggcctccaaat accccaagga 3601 ctgtagtgag gtcgagcgca cctacctgaa aaccaaatcaagctccccta gagacaagat 3661 ctacactata gatggtgaga aggagcctgg tttccacttagatccacccc agtttgttga 3721 aaatgtgacc ctgcccgaga acgtggactt cccggacccctaccaggatc ccagtgaaaa 3781 cttccgcaag ggggactcca cgctgccaat gaaccggaaccccttgcata atgaagaggg 3841 gctttccaac aacgaccagt ataaactcta ctccaagcacttcaccttga aagacaaggg 3901 ttccccgcac agtgagacca gcgagcgata ccggcagaactccacgcact gcagaagctg 3961 cctttccaac atgcccacct attcaggcca cttcaccatgaggtccccct tcaagtgcga 4021 tgcctgcctg cggatgggga acctctatga catcgatgaagaccagatgc ttcaggagac 4081 aggtaaccca gccaccgggg agcaggtcta ccagcaggactgggcacaga acaatgccct 4141 tcaattacaa aagaacaagc taaggattag ccgtcagcattcctacgata acattgtcga 4201 caaacctagg gagctagacc ttagcaggcc ctcccggagcataagcctca aggacaggga 4261 acggcttctg gagggaaatt tttacggcag cctgtttagtgtcccctcaa gcaaactctc 4321 ggggaaaaaa agctcccttt tcccccaagg tctggaggacagcaagagga gcaagtctct 4381 cttgccagac cacacctccg ataacccttt cctccactcccacagggatg accaacgctt 4441 ggttattggg agatgcccct cggaccctta caaacactcgttgccatccc aggcggtgaa 4501 tgacagctat cttcggtcgt ccttgaggtc aacggcatcgtactgttcca gggacagtcg 4561 gggccacaat gatgtgtata tttcggagca tgttatgccttatgctgcaa ataagaataa 4621 tatgtactct acccccaggg ttttaaattc ctgcagcaatagacgcgtgt acaagaaaat 4681 gcctagtatc gaatctgatg tttaaaaatc ttccattaatgttttatcta tagggaaata 4741 cacgtaatgg ccaatgttct ggagggtaaa tgttggatgtccaatagtgc cctgctaaga 4801 ggaagaagat gtagggaggt attttgttgt tgttgttgttggctcttttg cacacggctt 4861 catgccataa tcttccactc aaggaatctt gtgaggtgtgtgctgagcat ggcagacacc 4921 agataggtga gtccttaacc aaaaataact aactacataagggcaagtct ccgggacatg 4981 cctactgggt atgttggcaa taatgatgca ttggatgccaatggtgatgt tatgatttcc 5041 tatattccaa attccattaa ggtcagccca ccatgtaattttctcatcag aaatgcctaa 5101 tggtttctct aatacagaat aagcaatatg gtgtgcatgtaaacctgaca cagacaaaat 5161 aaaaacagtt aagaatgcat ctgcactgta gtcggatttgaacatgtgca agagattagg 5221 aagtttggct cgtaacagtt tcagctttct tgttatgccttccatcacag cccaggctca 5281 ccccaagaac tccaggctcc cctaaagaat agcaaatcagtgtgttcgtg atgactgtgc 5341 taccttcatt atagttcatt tccaagacac atctggagccaaaggcccga gggaccctca 5401 ggtggggaga gctacaggaa tctctttgga tgttgatgtgtgtttctctc taccctcggc 5461 ttcgatggtc ttgttcagag ctgcataaac taacacatttatgtctccga gatctaagtg 5521 tggatcttct gtctgtgaca cagtggccat tgtagtttatcccgaagacg cctatgtacg 5581 taagtttgca tttcctccct tctggtgatg actcagggttgtatagtatc tgttacccct 5641 tccctcccag agtaaccata actcgttccg tttccaaacagccatggtgg tgtccaatta 5701 gctgtgtatc gctcttccca gagttgttaa tgtggtgacatgcaccaaca gccgtatgtg 5761 tactgtgatc tgtaagaagt acaatgccat ctgtctgccgaaggctagca tggttttagg 5821 tttatcttcc ttcacatcca gaaattctgt tggacactcacttccacccc aaactcctca 5881 aatcaaaagc cttcaaaaca cgaggcactc ttggatctaccctgagtatc ctccaaactg 5941 tggatacagt ttagtgagac aagcaatttc tcccttctgagttattctct ctgttggtgg 6001 caaaccactt catagcacca acagagatgt aggaaaaattcctcaaagta tttgtcattt 6061 ctgagtcgcc tgcattatcc cattcttatt ctcctcaaacctgtgcatat atgacatgaa 6121 atgatatcca tttttttttt aagttagaaa cagagaggggaatacttatg catggggagc 6181 ctgttagcac agtgcctgcc acaaaaacaa gtgcccccgacaagatagtt gctatgttat 6241 gacactttct cagatcagga ttttctagtt taaaaattaaatatcataaa acg

[0144] SEQ ID NO:6. shows the oligonucleotide sequence of theauto-antigenic region of the N-terminal domain of the NR2A subunit, asfollows:

[0145] SEQ ID NO:6

[0146] N-terminal nucleotide sequence 371 ccggcgccga gcgcggcggcggagaagggt ccccccgcgc taaatattgc 421 ggtgatgctg ggtcacagcc acgacgtgacagagcgcgaa cttcgaacac tgtggggccc 481 cgagcaggcg gcggggctgc ccctggacgtgaacgtggta gctctgctga tgaaccgcac 541 cgaccccaag agcctcatca cgcacgtgtgcgacctcatg tccggggcac gcatccacgg 601 cctcgtgttt ggggacgaca cggaccaggaggccgtagcc cagatgctgg attttatctc 661 ctcccacacc ttcgtcccca tcttgggcattcatgggggc gcatctatga tcatggctga 721 caaggatccg acgtctacct tcttccagtttggagcgtcc atccagcagc aagccacggt 781 catgctgaag atcatgcagg attatgactggcatgtcttc tcoctggtga ccactatctt 841 ccctggctac agggaattca tcagcttcgtcaagaccaca gtggacaaca gctttgtggg 901 ctgggacatg cagaatgtga tcacactggacacttccttt gaggatgcaa agacacaagt 961 ccagctgaag aagatccact cttctgtcatcttgctctac tgttccaaag acgaggctgt 1021 tctcattctg agtgaggccc gctcccttggcctcaccggg tatgatttct tctggattgt 1081 ccccagcttg gtctctggga acacggagctcatcccaaaa gagtttccat cgggactcat 1141 ttctgtctcc tacgatgact gggactacagcctggaggcg agagtgaggg acggcattgg 1201 catcctaacc accgctgcat cttctatgctggagaagttc tcctacatcc ccgaggccaa 1261 ggccagctgc tacgggcaga tggagaggccagaggtcccg atgcacacct tgcacccatt 1321 tatggtcaat gttacatggg atggcaaagacttatccttc actgaggaag gctaccaggt 1381 gcaccccagg ctggtggtga ttgtgctgaacaaagaccgg gaatgggaaa aggtgggcaa 1441 gtgggagaac catacgctga gcctgaggcacgccgtgtgg cccaggtaca agtccttctc 1501 cgactgtgag ccggatgaca accatctcagcatcgtcacc ctggaggagg ccccattcgt 1561 catcgtggaa gacatagacc ccctgaccgagacgtgtgtg aggaacaccg tgccatgtcg 1621 gaagttcgtc aaaatcaaca attcaaccaatgaggggatg aatgtgaaga aatgctgcaa 1681 ggggttctgc attgatattc tgaagaagctttccagaact gtgaagttta cttacgacct 1741 ctatctggtg accaatggga agcatggcaagaaagttaac aatgtgtgga atggaatgat 1801 cggtgaagtg gtctatcaac gggcagtcatggcagttggc tcgctcacca tcaatgagga 1861 acgttctgaa gtggtggact tctctgtgccctttgtggaa acgggaatca gtgtcatggt 1921 ttcaagaagt aatggcaccg tctcaccttctgcttttcta gaaccattca gcgcctct

[0147] SEQ ID NO:7 shows a 62 oligonucleotide fragment target, asfollows:

[0148] SEQ ID NO:7

[0149] atggaatgatcggtgaagtggtctatcaacgggcagtcatggcagttggctcgctcaccatc

[0150] SEQ ID NO:8 shows one oligonucleotide primer, as follows:

[0151] SEQ ID NO:8

[0152] agcatggcaagaaagttaaca

[0153] SEQ ID NO:9 shows a second oligonucleotide primer, as follows:

[0154] SEQ ID NO:9

[0155] acgttctgaagtggtggactt

[0156] SEQ ID NO: 10. shows the full-length amino acid sequence of themature NR2B receptor subunit, as follows:

[0157] PEPTIDE

[0158] Homo sapiens glutamate receptor, ionotropic, N-methyl D-aspartate2B

[0159] Biochim. Biophys. Acta 1260:105-108(1995).

[0160] sequence NME2_HUMAN (Q13224)       1      11        21        31      41    1   MKPRAECCSPKFWLVLAVLA VSGSRARSQK SPPSIGIAVI         51       LVGTSDEVAI KDAHEKDDFH 60  61   HLSVVPRVEL VAMNETDPKS IITRICDLMS DRKIQGVVFA       DDTDQEAIAQ ILDFISAQTL 120 121   TPILGIHGGS SMIMADKDES SMFFQFGPSI EQQASVMLNI       MEEYDWYIFS IVTTYFPGYQ 180 181   DFVNKIRSTI ENSFVGWELE EVLLLDMSLD DGDSKIQNQL       KKLQSPIILL YCTKEEATYI 240 241   FEVANSVGLT GYGYTWIVPS LVAGDTDTVP AEFPTGLISV       SYDEWDYGLP ARVRDGIAII 300 301   TTAASDMLSE HSFIPEPKSS CYNTHEKRIY QSNMLNRYLI       NVTFEGRNLS FSEDGYQMHP 360 361   KLVIILLNKE RKWERVGKWK DKSLQMKYYV WPRMCPETEE       QEDDHLSIVT LEEAPFVIVE 420 421   SVDPLSGTCM RNTVPCQKRI VTENKTDEEP GYIKKCCKGF       CIDILKKISK SVKFTYDLYL 480 481   VTNGKHGKKI NGTWNGMIGE VVMKRAYMAV GSLTINEERS       EVVDFSVPFI ETGISVMVSR 540 541   SNGTVSPSAF LEPFSADVWV MMFVMLLIVS AVAVFVFEYF       SPVGYNRCLA DGREPGGPSF 600 601   TIGKAIWLLW GLVFNNSVPV QNPKGTTSKI MVSVWAFFAV       IFLASYTANL AAFMIQEEYV 660 661   DQVSGLSDKK FQRPNDFSPP FRFGTVPNGS TERNIRNNYA       EMHAYMGKFN QRGVDDALLS 720 721   LKTGKLDAFI YDAAVLNYMA GRDEGCKLVT IGSGKVFAST       GYGIAIQKDS GWKRQVDLAI 780 781   LQLFGDGEME ELEALWLTGI CHNEKNEVMS SQLDIDNMAG       VFYMLGAAMA LSLITFICEH 840 841   LFYWQFRHCF MGVCSGKPGM VFSISRGIYS CIHGVAIEER       QSVMNSPTAT MNNTHSNILR 900 901   LLRTAKNMAN LSGVNGSPQS ALDFIRRESS VYDJSEHRRS       FTHSDCKSYN NPPCEENLFS 960 961   DYISEVFRTF GNLQLKDSNV YQDHYHHHHR PHSIGSASSI       DGLYDCDNPP FTTQSRSISK 10201021   KPLDIGLPSS KHSQLSDLYG KFSFKSDRYS GHDDLIRSDV       SDISTHTVTY GNIEGNAAKR 10801081   RKQQYKDSLK KRPASAKSRR EFDEIELAYR RRPPRSPDHK       RYFRDKEGLR DFYLDQFRTK 11401141   ENSPHWBHVD LTDLYKERSD DFKRDSVSGG GPCTNRSHIK       HGTGDKHGVV SGVPAPWEKN 12001201   LTNVEWEDRS GGNFCRSCPS KLHNYSTTVT GQNSGRQACI       RCEACKKAGN LYDISEDNSL 12601261   QELDQPAAPV AVTSNASTTK YPQSPTNSKA QKKNRNKLRR       QHSYDTFVDL QKEEAALAPR 13201321   SVSLKDKGRF MDGSPYAHMF EMSAGESTFA NNKSSVPTAG       HHHHNNPGGG YMLSKSLYPD 13801381   RVTQNPFIPT FGDDQCLLHG SKSYFFRQPT VAGASKARPD       FRALVTNXPV VSALHGAVPA 14401441   RFQKDICIGN QSNPCVPNNK NPRAFNGSSN GHVYEKLSSI

[0161] SEQ ID NO: 11. shows the amino acid sequence of theauto-antigenic region of the N-terminal domain of the NR2B subunit, asfollows:

[0162] SEQ ID NO: 11

[0163] Homo sapiens                      RSQK SPPSIGIAVI LVGTSDEVAI                KDAHEKDDFH 60 61 HLSVVPRVEL VAMNETDPKS IITRICDLMSDRKIQGVVFA DDTDQEAIAQ ILDFISAQTL 120 121 TPILGIHGGS SMIMADKDESSMFFQFGPSI EQQASVMLNI MEEYDWYIFS IVTTYFPGYQ 180 181 DFVNKJRSTIENSFVGWELE EVLLLDMSLD DGDSKIQNQL KKLQSPIILL YCTKEEATYI 240 241FEVANSVGLT GYGYTWIVPS LVAGDTDTVP AEFPTGLISV SYDEWDYGLP ARVRDGIAII 300301 TTAASDMLSE HSFIPEPKSS CYNTHEKRIY QSNMLNRYLI NVTFEGRNLS FSEDGYQMHP360 361 KLVIILLNKE RKWERVGKWK DKSLQMKYYV WPRMCPETEE QEDDHLSIVTLEEAPFVIVE 420 421 SVDPLSGTCM RNTVPCQKRI VTENKTDEEP GYIKKCCKGFCIDILKKISK SVKFTYDLYL 480 481 VTNGKHGKKI NGTWNGMIGE VVMKRAYMAVGSLTTNEERS EVVDFSVPFI ETGISVMVSR 540 541 SNGTVSPSAF LEPFSAD

[0164] SEQ ID NO: 12; shows a 20 amino acid antigenic peptide fragmentof the NR2B subunit, as follows:

[0165] SEQ ID NO:12

[0166] Homo sapiens

[0167] GYIKKCCKGF CIDILKKISK

[0168] SEQ ID NO: 13 shows a 21 amino acid sequence of an antigenicfragment of the NR2B subunit modified by an N-terminal Cys forattachment to a carrier protein, as follows:

[0169] SEQ ID NO:13.

[0170] Artificial Sequence (21 aminoacids)

[0171] CGYIKKCCKGF CIDILKKISK

[0172] FULL

[0173] BASE COUNT ORIGIN

[0174] SEQ ID NO: 14 shows the oligonucleotide position numbering usedthroughout in reference to NR2B oligonucleotide sequences, as follows:

[0175] SEQ. NO. 14 Homo sapiens glutamate receptor, ionotropic, N-methylD-aspartate 2B mRNA 1 ttgaatttgc atctcttcaa gacacaagat taaaacaaaatttacgctaa attggatttt 61 aaattatctt ccgttcattt atccttcgtc tttcttatgtggatatgcaa gcgagaagaa 121 gggactggac attcccaaca tgctcactcc cttaatctgtccgtctagag gtttggcttc 181 tacaaaccaa gggagtcgac gagttgaaga tgaagcccagagcggagtgc tgttctccca 241 agttctggtt ggtgttggcc gtcctggccg tgtcaggcagcagagctcgt tctcagaaga 301 gcccccccag cattggcatt gctgtcatcc tcgtgggcacttccgacgag gtggccatca 361 aggatgccca cgagaaagat gatttccacc atctctccgtggtaccccgg gtggaactgg 421 tagccatgaa tgagaccgac ccaaagagca tcatcacccgcatctgtgat ctcatgtctg 481 accggaagat ccagggggtg gtgtttgctg atgacacagaccaggaagcc atcgcccaga 541 tcctcgattt catttcagca cagactctca ccccgatcctgggcatccac gggggctcct 601 ctatgataat ggcagataag gatgaatcct ccatgttcttccagtttggc ccatcaattg 661 aacagcaagc ttccgtaatg ctcaacatca tggaagaatatgactggtac atcttttcta 721 tcgtcaccac ctatttccct ggctaccagg actttgtaaacaagatccgc agcaccattg 781 agaatagctt tgtgggctgg gagctagagg aggtcctcctactggacatg tccctggacg 841 atggagattc taagatccag aatcagctca agaaacttcaaagccccatc attcttcttt 901 actgtaccaa ggaagaagcc acctacatct ttgaagtggccaactcagta gggctgactg 961 gctatggcta cacgtggatc gtgcccagtc tggtggcaggggatacagac acagtgcctg 1021 cggagttccc cactgggctc atctctgtat catatgatgaatgggactat ggcctccccg 1081 ccagagtgag agatggaatt gccataatca ccactgctgcttctgacatg ctgtctgagc 1141 acagcttcat ccctgagccc aaaagcagtt gttacaacacccacgagaag agaatctacc 1201 agtccaatat gctaaatagg tatctgatca atgtcacttttgaggggagg aatttgtcct 1261 tcagtgaaga tggctaccag atgcacccga aactggtgataattcttctg aacaaggaga 1321 ggaagtggga aagggtgggg aagtggaaag acaagtccctgcagatgaag tactatgtgt 1381 ggccccgaat gtgtccagag actgaagagc aggaggatgaccatctgagc attgtgaccc 1441 tggaggaggc accatttgtc attgtggaaa gtgtggaccctctgagtgga acctgcatga 1501 ggaacacagt cccctgccaa aaacgcatag tcactgagaataaaacagac gaggagccgg 1561gttacatcaa`aaaatgctgc`aaggggttct`gtattgacat`ccttaagaaa`atttctaaat 1621ctgtgaagtt cacctatgac ctttacctgg ttaccaatgg caagcatggg aagaaaatca 1681atggaacctg gaatggtatg attggagagg tggtcatgaa gagggcctac atggcagtgg 1741gctcactcac catcaatgag gaacgatcgg aggtggtcga cttctctgtg cccttcatag 1801agacaggcat cagtgtcatg gtgtcacgca gcaatgggac tgtctcacct tctgccttct 1861tagagccatt cagcgctgac gtatgggtga tgatgtttgt gatgctgctc atcgtctcag 1921ccgtggctgt ctttgtcttt gagtacttca gccctgtggg ttataacagg tgcctcgctg 1981atggcagaga gcctggtgga ccctctttca ccatcggcaa agctatttgg ttgctctggg 2041gtctggtgtt taacaactcc gtacctgtgc agaacccaaa ggggaccacc tccaagatca 2101tggtgtcagt gtgggccttc tttgctgtca tcttcctggc cagctacact gccaacttag 2161ctgccttcat gatccaagag gaatatgtgg accaggtttc tggcctgagc gacaaaaagt 2221tccagagacc taatgacttc tcaccccctt tccgctttgg gaccgtgccc aacggcagca 2281cagagagaaa tattcgcaat aactatgcag aaatgcatgc ctacatggga aagttcaacc 2341agaggggtgt agatgatgca ttgctctccc tgaaaacagg gaaactggat gccttcatct 2401atgatgcagc agtgctgaac tatatggcag gcagagatga aggctgcaag ctggtgacca 2461ttggcagtgg gaaggtcttt gcttccactg gctatggcat tgccatccaa aaagattctg 2521ggtggaagcg ccaggtggac cttgctatcc tgcagctctt tggagatggg gagatggaag 2581aactggaagc tctctggctc actggcattt gtcacaatga gaagaatgag gtcatgagca 2641gccagctgga cattgacaac atggcagggg tcttctacat gttgggggcg gccatggctc 2701tcagcctcat caccttcatc tgcgaacacc ttttctattg gcagttccga cattgcttta 2761tgggtgtctg ttctggcaag cctggcatgg tcttctccat cagcagaggt atctacagct 2821gcatccatgg ggtggcgatc gaggagcgcc agtctgtaat gaactccccc accgcaacca 2881tgaacaacac acactccaac atcctgcgcc tgctgcgcac ggccaagaac atggctaacc 2941tgtctggtgt gaatggctca ccgcagagcg ccctggactt catccgacgg gagtcatccg 3001tctatgacat ctcagagcac cgccgcagct tcacgcattc tgactgcaaa tcctacaaca 3061acccgccctg tgaggagaac ctcttcagtg actacatcag tgaggtagag agaacgttcg 3121ggaacctgca gctgaaggac agcaacgtgt accaagatca ctaccaccat caccaccggc 3181cccatagtat tggcagtgcc agctccatcg atgggctcta cgactgtgac aacccaccct 3241tcaccaccca gtccaggtcc atcagcaaga agcccctgga catcggcctc ccctcctcca 3301agcacagcca gctcagtgac ctgtacggca aattctcctt caagagcgac cgctacagtg 3361gccacgacga cttgatccgc tccgatgtct ctgacatctc aacccacacc gtcacctatg 3421ggaacatcga gggcaatgcc gccaagaggc gtaagcagca atataaggac agcctgaaga 3481agcggcctgc ctcggccaag tcccgcaggg agtttgacga gatcgagctg gcctaccgtc 3541gccgaccgcc ccgctcccct gaccacaagc gctacttcag ggacaaggaa gggctacggg 3601acttctacct ggaccagttc cgaacaaagg agaactcacc ccactgggag cacgtagacc 3661tgaccgacat ctacaaggag cggagtgatg actttaagcg cgactccatc agcggaggag 3721ggccctgtac caacaggtct cacatcaagc acgggacggg cgacaaacac ggcgtggtca 3781gcggggtacc tgcaccttgg gagaagaacc tgaccaacgt ggagtgggag gaccggtccg 3841ggggcaactt ctgccgcagc tgtccctcca agctgcacaa ctactccacg acggtgacgg 3901gtcagaactc gggcaggcag gcgtgcatcc ggtgtgaggc ttgcaagaaa gcaggcaacc 3961tgtatgacat cagtgaggac aactccctgc aggaactgga ccagccggct gccccagtgg 4021cggtgacgtc aaacgcctcc accactaagt accctcagag cccgactaat tccaaggccc 4081agaagaagaa ccggaacaaa ctgcgccggc agcactccta cgacaccttc gtggacctgc 4141agaaggaaga agccgccctg gccccgcgca gcgtaagcct gaaagacaag ggccgattca 4201tggatgggag cccctacgcc cacatgtttg agatgtcagc tggcgagagc acctttgcca 4261acaacaagtc ctcagtgccc actgccggac atcaccacca caacaacccc ggcggcgggt 4321acatgctcag caagtcgctc taccctgacc gggtcacgca aaaccctttc atccccactt 4381ttggggacga ccagtgcttg ctccatggca gcaaatccta cttcttcagg cagcccacgg 4441tggcgggggc gtcgaaagcc aggccggact tccgggccct tgtcaccaac aagccggtgg 4501tctcggccct tcatggggcc gtgccagccc gtttccagaa ggacatctgt atagggaacc 4561agtccaaccc ctgtgtgcct aacaacaaaa accccagggc tttcaatggc tccagcaatg 4621ggcatgttta tgagaaactt tctagtattg agtctgatgt ctgagtgagg gaacagagag 4681gttaaggtgg gtacgggagg gtaaggctgt gggtcgcgtg atgcgcatgt cacggagggt 4741gacgggggtg aacttggttc ccatttgctc ctttcttgtt ttaatttatt tatgggatcc 4801tggagttctg gttcctactg ggggcaaccc tggtgaccag caccatctct cctccttttc 4861acagttctct ccttcttccc cccgctgtca gccattcctg ttcccatgag atgatgccat 4921gggccctctc agcaggggag ggtagagcgg agaaaggaag ggctgcatgc gggcttcctc 4981ctggtgtgga agagctcctt gatatcctct ttgagtgaag ctgggagaac caaaaagagg 5041ctatgtgagc acaaaggtag cttttcccaa actgatcttt tcatttaggt gaggaagcaa 5101aagcatctat gtgagaccat ttagcacact gcttgtgaaa ggaaagaggc tctggctaaa 5161ttcatgctgc ttagatgaca tctgtctagg aatcatgtgc caagcagagg ttgggaggcc 5221atttgtgttt atatataagc ccaaaaatgc ttgcttcaac cccatgagac tcgatagtgg 5281tggtgaacag aacccaaggt cattggtggc agagtggatt cttgaacaaa ctggaaagta 5341cgttatgata gtgtcccccg gtgccttggg gacaagagca ggtggattgt gcgtgcatgt 5401gtgttcatgc acacttgcac ccatgtgtag tcaggtgcct caagagaagg caaccttgac 5461tctttctatt gtttctttca atatccccaa gcagtgtgat tgtttggctt atatacagac 5521agagatggcc atgtattacc tgaattttgg ctgtgtctcc cttcatcctt ctggaataag 5581gagaatgaaa attcttgata aagaagattc tgtggtctaa acaaaaaaag gcggtgagca 5641atcctgcaag aacaaggtac ataaacaagt cctcagtggt tggcaattgt ttcaaccagt 5701ttgaaccaag aactttccag gaaggctaaa gggaaaccga attttcacag ccatgattct 5761tttgcccaca cttgggagca aaagattcta caaagctctt ttgagcattt agactctcga 5821ctggccaagg tttggggaag aacgaagcca cctttgaaga agtaaggagt cgtgtatggt 5881agggtaagtg agagaggggg atgtttccaa tgctttgatc ccttcttact taacctgaag 5941ctagacgagc aggcttcttc cccccaaaac tgattacaac tgctacagag cagacagtta 6001agagaaatga gcttgacctt taagagaaat gagctgcact ccatgagtgc agctctggag 6061gtacgaaaag aggggaagag acttggaaat gggagacggg ggcagagagg gaccctccac 6121cacctctttg ggcctggctc cctgggaatg tgacttgagc ccagagtgaa cactcttggt 6181agaagccctt ctaccttcct gcaacacctt gtttccctct cagattgtac cattgag

[0176] SEQ ID NO:15 shows a 60 oligonucleotide fragment target, asfollows:

[0177] SEQ ID NO:15

[0178] g gttacatcaa aaaatgctgc aaggggttct gtattgacat ccftaagaaaatttctaaa

[0179] SEQ ID NO:16 shows one oligonucleotide primers (21 nucleotides),as follows:

[0180] SEQ ID NO:16

[0181] tcactgagaa taaaacagac g

[0182] SEQ ID NO:17 shows one oligonucleotide primers (21 nucleotides),as follows:

[0183] SEQ ID NO:17

[0184] t cacctatgac ctttacctgg

EXAMPLES

[0185] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow the compounds claimed herein are made and evaluated, and areintended to be purely exemplary of the invention and are not intended tolimit the scope of what the inventors regard as their invention. Effortshave been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at room temperature, and pressure is at ornear atmospheric.

Example 1 Preparation of Polyclonal Antibodies (IgG) to Glutamate andHomocysteine

[0186] Glutamate (polyglutamate, 10 aminoacids) or homocysteine(polyhomocysteine, 10 aminoacids) alone will not generate antibodieswhen injected into an animal. Therefore, polyglutamate andpolyhomocysteine were conjugated with human serum albumin for theimmunization to obtain polyclonal antibodies. For glutaraldehydeconjugation, polyglutamate or homocysteine (10 mg) and 40 mg bovineserum albumin (BSA, Sigma, St. Louis, Mo.) were incubated for 2 hr atroom temperature in 4 ml of PBS containing 5% glutaraldehyde. Thereaction was stopped by adding glycine to a final concentration of 0.2M, and the conjugate was dialyzed against PBS.

[0187] Rabbits were given initial injections of 1 mg of conjugatedglutamate (polyglutamate) or homocysteine (polyhomocysteine) in completeFreund's and subsequent increased doses of injections (2 mg) inincomplete Freund's adjuvant at successive 2 week intervals. Allinjections were given subcutaneously. The immunization period lasted for110 days. Antibodies (IgG) were affinity purified according to standardprocedures (Warr, G. W., Purification of antibodies, In: Antibody as aTool, Eds., Marchalonis, J. J., and G. W. Warr, J. Wiley, UK, pp. 59-96(1982)) and were shown to be selective for glutamate or homocysteine byELISA assay.

Example 2 Preparation of Polyclonal Antibodies (IgG) to NR2A ReceptorPeptide

[0188] Using computer analysis of the hydrophobicity profile of humanNR2A and NR2B NMDA receptors to predict the antigenic determinants inthe protein structure, we selected fragments corresponding to theN-terminal sequence of human NR2A and NR2B receptor peptides forsynthesis. The fragments corresponded to the N-terminal sequence of theNR2A and NR2B receptors, represented by SEQ ID NO: 1 and SEQ ID NO: 2for the NR2A and NR2B receptors, respectively. The peptide fragmentswere reproduced using solid-phase synthesis, and had a purity rangingfrom 90% to 98%. The peptide sequences were verified by amino acidanalysis after acid hydrolysis. A mixture of NR2A and NR2B peptides(1:1) was conjugated with human serum albumin for the immunization toobtain polyclonal antibodies. For glutaraldehyde conjugation, 10 mg ofthe mixture of peptides and 40 mg human serum albumin (Sigma, St. Louis,Mo.) were incubated for 1.45 hr at room temperature in 4 ml of PBScontaining 5% glutaraldehyde. The reaction was stopped by adding glycineto a final concentration of 0.2 M, and the conjugate was dialyzedagainst PBS.

[0189] Rabbit polyclonal antibodies were raised against the NR2A-Bpeptides. Rabbits were given initial injections of 1 mg of conjugatedpeptides in complete Freund's adjuvant and subsequent injections (0.5mg) in incomplete Freund's adjuvant at successive 2 week intervals.Antibodies were affinity purified according to standard procedures(Warr, G. W., Purification of antibodies, In: Antibody as a Tool, Eds.,Marchalonis, J. J., and G. W. Warr, J. Wiley, UK, pp. 59-96 (1982)) andwere shown to be selective for NR2A and NR2B NMDA receptors using anELISA assay.

Example 3 Preparation of Latex Beads Containing Biomarker Antibodies

[0190] Three different sensitized latex beads containing IgG againstglutamate, homocysteine and NR2A-B receptor peptides were prepared usingtwo types of blue polystyrene latex beads (diameter, 0.25 and 0.4 μM;Sigma, St.Louis, Mo.) as follows. A 1% suspension of latex beads in 50mM PBS (1 ml, pH 7.0) was mixed with an equal volume of correspondingIgG (2 mg/ml) and incubated on a shaker at room temperature for 2 hours.The mixture was then washed twice with PBS by centrifugation at 9,500 gfor 5 min. The pellet was suspended in PBS containing 1% BSA overnightat 4° C. After being washed twice with PBS, the sensitized latex beadswere resuspended in latex diluent (50 mM PBS with 1% BSA) at aconcentration of 0.4% and stored at 4° C. until used.

[0191] Preliminary experiments with latex agglutination (LA) alone wereperformed to identify problems and to select the most desirable latexparticle size. Two types of commercial latex beads were coated withantibodies at various concentrations. Tests were initially performedwith the corresponding aminoacid or NR2A and NR2B receptor peptides ascontrols. Particle size and IgG concentration were found to be theprimary factors affect the sensitivity of the test. The most desirableparticle size was found to be 0.25 μm (blue latex) because particles ofthis size agglutinated each aminoacid and peptide specifically. HigherIgG concentrations showed higher sensitivities. Using blue latex beadcoated with 2 mg of IgG per ml, agglutination could be observed within30 min.

Example 4 Latex Agglutination Analysis of Blood Serum Specimens

[0192] Blood samples (5 ml) were collected using standard venipunctureclinical protocol, from patients with TIA, stroke and brain injury(n=30) and examined at the laboratory of CIS Biotech, Inc. in Atlanta(Ga., USA). None of the patients had been treated with anticoagulants,and serum samples were obtained from the clotted blood. All specimenswere free of visible lipids, white blood cells, platelets, fibrin, mucusor other contaminants that could cause “false positive” reactions.Platelets, white blood cells, mucus and fibrin were removed bycentrifugation. Lipids were removed by filtration.

[0193] Specimens to be tested within 72 hours after collection werestored at 2-8° C. For longer storage periods, −20° C. or colder isrecommended.

[0194] The semi-quantitative analysis of glutamate, homocysteine andNR2A-B receptor peptides in the serum samples is basically a three stepprocess: serum sample dilution, reaction of latex beads with serumsamples, and product analysis.

[0195] In previous experiments serial dilutions of the serum samplesfrom 1:4 to 1:64 in saline containing 4% glycerol for betteragglutination were performed. The highest dilution in whichagglutination was observed corresponded to the sample titer.

[0196] Two 25 μl aliquots of coated latex beads containing thecorresponding IgG were layered on a double-concave slide (Fisher Sci.,Norcross, Ga.), one with 25 μl of the serum sample in serial dilution tobe tested and one with 25 μl of PBS as a negative control. After gentlemixing with vortex, agglutination was judged macroscopically against adark background. A negative reaction corresponded to a homogeneouslactescent background with no agglutination; a positive reactioncorresponded to a clearly visible agglutination against the blackbackround and weakly visible agglutination on a slightly lactescentbackground.

[0197] The highest dilution at which agglutination occurs gives thetiter of the sample. To obtain the approximate titer in μg/ml we usedthe following calculation:

Titer μg/ml=A×D

[0198] where A is the test sensitivity, and D is the highest dilution atwhich agglutination occurs.

Example 5 Description of Patients

[0199] Patients observed in trials (n=68) included 9 with pre-stroke, 9with TIA (mean age 52.0±3.0), 31 with acute ischemic stroke (mean age54.7±1.4) and 11 with mild brain injury (mean age 53.0±4.4). Clinicalevaluation of patients by neuroimaging (CT, MRI, arteriography, Dopplerultrasonography, EEG), detailed physical and neurologic examination andlaboratory tests was performed. Patients with TIA were characterized bycontra lateral weakness, dysphasia, transient blurring of vision orblindness, abnormal pulsation of the common carotid arteries,microemboli confined to the ipsilateral retina. Untreated patients withpre-stroke demonstrated altered state of consciousness, severe headache,nausea and vomiting, visual disturbances, and focal neurologicaldeficit, with some patients experiencing seizures.

[0200] The N-Score rating scale reported in “MCA Infarction” (Orgogozo,1986) was used for evaluating the neurologic deficit in patients withacute cerebral stroke. The total score of acute cerebral stroke clinicalmanifestation differentiated severe patients (n=9, 11-35 scores) frompatients with mild (n—12, 36-55 scores) and moderate patients (n=10,60-90 scores). Most patients with acute cerebral ischaemia (61.3%)suffered ischemia in the carotid artery of left hemisphere. Arterialhypertension and cerebral atherosclerosis etiologically corresponded inall patients.

[0201] The patients with ischemia were divided into groups based on thedifferences between TIA, pre-stroke and acute ischemic pathogenicmechanisms. The clinical diagnosis was established on the basis ofroutine observations which included detailed neurological examinationand neuroimaging. Groups of TIA (n=9) and pre-stroke patients withchronic cerebral blood insufficiency (n=9) were identified byneurophysiological investigations.

Example 6 Detection of Glutamate and Homocysteine in the Blood ofPatients

[0202] Glutamate and homocysteine content were measured by standard highperformance liquid chromatography (HPLC) according to methods described(Perry I. J., Refsum H., Morris R. W., Ebrahim S. B., Ueland P. M.,Shaper A. G. Lancet. 1995, 346:1395-1398; Yamamoto T., Rossi S., StiefelM., Doppenberg E., Zauner A., Bullock R., Marmarou A. ActaNeurochir.Suppl. 1999, 75: 17-19). The limits of the normal range were165.0 μmol/L for glutamate (Table 1) and 8.0 μmol/L for homocysteine(Table 2). Elevated glutamate and homocysteine amounts were detected inthe blood of patients with acute stroke. However, approximately 66% ofthese patients had additional risk factors indicative of atheroscleroticprocesses such as high cholesterol and LDL levels (Denisenko T. V.,Skuliabin D., Gromov I., Cherkas Yi., Iluchina A., Dambinova S. A.,1998. Vopr. Med. Khimii. 44, 584-590, in Russian).

[0203] Abnormal glutamate and homocysteine plasma concentrations wereobserved more frequently in patients with TIA than in patients withacute stroke. The positive predictive efficiency of plasma glutamate forTIA patients was 56%. The positive predictive efficiency of plasmahomocysteine for TIA patients was 66%. Baseline concentrations forglutamate and homocysteine are 160 mmol/L and 10 umol/L, respectively.Routine treatment for TIA was found to consistently decrease theglutamate and homocysteine levels in the blood of patients (data notshown).

[0204] In patients with pre-stroke, slightly elevated levelshomocysteine were observed; levels of glutamate were unchanged (Tables1, 2). In patients with traumatic brain injury (TBI), glutamate levelswere observed that were nearly twice the glutamate levels in healthyindividuals; levels of homocysteine were up to 57% higher. TABLE 1Glutamate concentration in the blood of patients detected by HPLC HPLCpredictive value Total Glutamate Negative Positive Group N μmol/L N % N% Healthy individuals 28 165.0 ± 28.2 19 67.8  9 32.2 TIA  9 200.0 ±11.7  4 44.4  5 55.6 Pre-stroke  9 163.7 ± 10.4  5 55.6  4 44.4 Acutestroke 31 172.1 ± 20.6 13 41.9 18 58.1 TBI 11 305.0 ± 28.8  4 36.4  763.6

[0205] We also compared homocysteine concentrations in the blood ofpatients with TIA and pre-stroke to homocystein concentrations in theblood of patients who have had stroke onsets. We observed thathomocysteine content in the blood of patients depended on stage of thestroke, but that homocysteine concentration did not correlate with theseverity of the cerebral ischemia. A significant decrease inhomocysteine levels in patients with acute stroke was observed afteremergency therapy (data not shown).

[0206] Latex agglutination was also employed to detect TIA/strokebiomarkers in the blood serum of patients. The titer of plasma glutamatedetermined by latex agglutination was 3.34±0.25 in the group of healthyvolunteers. Homocysteine and glutamate trends observed using HPLC weresimilarly observed for different groups of patients observed by usingthe LA technique (Tables 3, 4). Thus, increased levels of glutamate andhomocysteine were similarly observed in the blood of patients with TIAand acute stroke using LA.

[0207] With respect to predictive efficiency, however, LA showed asurprising improvement over HPLC. For example, the LA method improvedthe positive predictive efficiency of patients with TIA and acute strokeon the basis of glutamate content to more than 63% (Tables 1, 3). Thenegative predictive value for healthy patients was similarly improvedwhen using the LA technique (Tables 3, 4). The predictive value of theLA technique in the group of patients with TBI was identical to thepredictive value using HPLC. TABLE 2 Homocysteine concentration in theblood of patients detected by HPLC HPLC predictive value TotalHomocysteine Negative Positive Group N μmol/L N % N % Healthyindividuals 28  8.0 ± 1.7 20 71.4 8 28.6 TIA  9 10.8 ± 1.3  3 33.3 666.4 Pre-stroke  9  9.0 ± 1.2  4 44.4 5 55.6 Acute stroke 31 11.5 ± 1.111 35.5 20  64.5 TBI 11 12.6 ± 2.1  4 36.4 7 63.6

[0208] TABLE 3 Detection of glutamate in the blood of patients by latexagglutination LA predictive value Total Glutamate Negative PositiveGroup N Titer N % N % Healthy individuals 28 3.34 ± 0.25 22 78.6 6 21.4TIA  9 4.52 ± 0.38  3 33.3 6 66.4 Pre-stroke  9 3.57 ± 0.32  4 44.4 555.6 Acute stroke 31 4.34 ± 0.47 11 35.5 20  64.5 TBI 11 5.12 ± 0.62  436.4 7 63.6

[0209] TABLE 4 Detection of homocysteine in the blood of patients bylatex agglutination LA predictive value Total Homocysteine NegativePositive Group N Titer N % N % Healthy individuals 28 2.23 ± 0.21 2175.0 7 25.0 TIA  9 3.95 ± 0.37  3 33.3 6 66.4 Pre-stroke  9 2.89 ± 0.12 4 44.4 5 55.6 Acute stroke 31 4.01 ± 0.41 10 32.3 21  67.7 TBI 11 4.74± 0.38  4 36.4 7 63.6

Example 7 The Detection of NR2A-B in the Blood of Patients

[0210] The excessive activation and damage of NMDA receptors is theresult of glutamate, aspartate and homocysteine neurotoxicity.Autoantibodies to have been detected in previous work in the blood ofpatients with TIA and pre-stroke, supporting our hypothesis thatcerebral ischemia causes neuronal damage and the appearance ofautoantibodies to NMDA receptor subunits (Gusev E. I., Skvortsova V. I.,Alekseev A. A., Izykenova G. A., Dambinova S. A. S. S Korsakov'sJ.Neurol.& Psych. 1996, 5:68-72; Dambinova S. A., Izykenova G. A. J.HighNervous Activity. 1997, 47: 439-446).

[0211] The titer of NR2A-B receptor peptides in the blood of healthyvolunteers determined by LA was 2.63±0.92. Using the LA technique, weobserved an increase in the test efficiency in the group of healthypersons up to 89% (Table 5). We also observed an improvement in thesensitivity of the LA test over ELISA. For example, patients withpre-stroke had slightly increased levels of NR2A-B receptor peptidesover healthy volunteers when tested by ELISA, but had nearly double thelevel of NR2A-B receptor peptides when measured by LA (Table 5, 6). Wedetected high levels of NR2A-B receptor peptides using both ELISA and LAin the blood of patients with TIA and acute stroke, and observedcomparable levels of predictive efficiency for each test.

[0212] Patients with TIA received routine treatment to improve braincirculation. Upon receiving treatment, NR2A-B levels decreased to levelscorresponding to those observed for the healthy individuals as thepatient's state normalized. As mentioned earlier, glutamate andhomocysteine contents also decreased during treatment, but it neverreached the levels observed in healthy individuals. TABLE 5 Detection ofNR2A-B receptor peptides in the blood of patients by latex agglutinationLA predictive value Total NR2A-B Negative Positive Group N Titer N % N %Healthy individuals 28 2.63 ± 0.92 25  89.3 3 10.7 TIA  9 7.34 ± 0.43 222.2 7 77.8 Pre-stroke  9 4.21 ± 0.26 2 22.2 7 77.8 Acute stroke 31 5.20± 1.71 4  9.7 27  87.1 TBI 11 3.99 ± 0.44 2 18.8 9 81.8

[0213] Completely different profiles of NR2A-B were revealed in theblood of patients with acute ischemic stroke. In the blood of patients(n=8) with severe cerebral ischaemia (30.4±3.2 Orgogozo scores) NR2A-Breceptor peptides titer was 4 times higher than that for control groupof healthy individuals. The peptides titer for patients with mild tomoderate ischemic stroke (n=22, 49-62 Orgogozo scores) was slightlyelevated in comparison with those with TIA. The tendency of slightdecreases in NR2A-B receptor peptide levels was observed to the end of30 days of patients' routine treatment, correlating with improvement inthe neurological state. TABLE 6 Detection of NR2A-B receptor peptides inthe blood of patients by ELISA ELISA assay results Total NR2A-B NegativePositive Group N Ng/ml N % N % Healthy individuals 28 18.2 ± 2.1 20 71.4 8 28.6 TIA  9 66.6 ± 4.1 2 22.2 7 77.8 Pre-stroke  9 23.7 ± 1.9 333.3 6 66.7 Acute stroke 31 73.4 ± 6.5 5 16.1 26  83.9 TBI 11 54.3 ± 4.93 27.3 8 72.7

[0214] It is necessary notice that efficiency of both laboratory assaysto detect the NR2A-B receptor peptides in the blood of patients withTIA/stroke and traumatic brain injury have been determined as 78 and 82%correspondingly.

[0215] The simultaneous detection of all brain damage biomarkers:glutamate, homocysteine and NR2A-B receptor peptides in the bloodpatients by latex agglutination allowed to diagnose rapidly TIA/strokewith efficiency up to 85-89%. The simultaneously increased levels of allbiomarkers in the blood reflect the neurological deficit and may be usedalso for prognosis of diseases outcome. The relation between thesebiomarkers is showing the degree of thromboembolic and neurotoxicityinvolvement in brain processes underlying the ischemia. That fact isvery important for choosing the strategy of emergency therapy in shorttime.

[0216] Using the latex agglutination technique allowed us significantlycut off the time of blood analysis from 3-8 hours in ELISA or HPLC to 30min in LA. This RMP semi-quantitative test demonstrated the fast, simplefor interpretation and reliable data.

Example 8 Identification of cDNA Sequence Encoding AntigenicDeterminants of NMDA Receptors

[0217] It was necessary to first determine the cDNA sequence coding theimmunological fragment of NMDA receptors responsible for the appearanceof autoantibodies appearance. To find the most active peptide fragmentof NMDA receptors a standard molecular biology procedure was used.Immunopositive phage GT11 containing cDNA coding NMDA receptors wasisolated from a human cDNA library using autoantibodies to NMDAreceptors isolated from blood samples of patients with severe cerebralischemia or polyclonal antibodies to the NR2A receptor. An E. Colibacterial system was employed to express the phage GT11 cDNA (600 bp).The expression product was transferred to a MBmp11 vector and arestriction map was constructed by use of a standard restrictases' kit.Three unique sites of the cDNA fragment (PstI, BamHI, and PsaI) wererevealed, and the 5′-3′ oligonucleotide sequence orientation using KpnI,BamHI and EcoR1 was deduced. The oligonucleotide (target cDNA) obtainedwas sequenced and compared to the sequence of the NR2A glutamatereceptor (SEQ ID NO: 5) from the NCBI library. The target cDNAcorresponded to the N-ternimal domain of the NR2A receptor (620 bp) ofSEQ ID NO: 6, namely SEQ ID NO: 7. Primers for this target nucleotidewere designed. All the oligonucleotides were prepared by thephosphoramidite method on an Applied Biosystem 394 synthesizer and werepurified by reverse-phase high-pressure liquid chromatography (HPLC).The oligonucleotides used for detection and capture were synthesizedwith an amine arm at the 5′ end.

Example 9 PCR Analysis of Blood Serum Specimens

[0218] Blood samples (5 ml collected by venipuncture) from patients withTIA and pre-stroke (n=30) were collected according to standard clinicalprotocol and examined at the Department of Neurology of Human BrainInstitute, St. Petersburg Russia. The blood specimens were used fortotal DNA isolation or applied on FTA paper circles.

[0219] The quantitative analysis of NR2A cDNA expression in the serumsamples is basically a three step process: Total DNA isolation andpurification from sera of individuals; specific cDNA coding NR2Areceptor amplification; and product analysis.

[0220] The total DNA isolated by DNAzol (Mol.Res.Center, Inc.,Cincinnati, Ohio) or bound on FTA blood staining collection cards (LifeTechnologies, Inc., Gathersburg, Md.) serves as a template for thepolymerase chain reaction (PCR). In the first variant, the PCR assayuses a set of specially designed primers (50 pmol), immobilized on solidmatrix of microplates and amplifies a specific cDNA sequence (620 bp)coding the NR2A glutamate receptor. In a second variant, the PCR assayuses a master ready-to-use buffer and amplifies cDNA bond on FTA paper.Following amplification, the quantity of a product is determined byenzyme or non-enzyme color reaction with a substrate.

[0221] Using the DNAzol reagent for DNA isolation, the whole blood ofeach individual (0.5 ml) was combined with 1 ml DNAzol (Mol.Res.Center,Inc., Cincinnati, Ohio) for 5 min at room temperature and lysed (MackeyK. et al. Mol.Biotechnol. 9: 1-5 (1997). The organic phase (0.4 ml) ofeach sample was transferred to a clean tube and 0.4 ml isopropanol wasadded. The mixture was incubated for 5 min at room temperature andcentrifugated at 6,000 g for 6 minutes. The pellet was washed in 0.5 mlDNAzol and centrifugated at the same conditions. The total DNA pelletwas mixed with 1 ml of 75% ethanol and centrifuged at 6,000 g for 5minutes. Then the DNA pellet was diluted in 200 l of 8 mM NaOH andincubated at room temperature for 5 min followed by vortexing. AlkalineDNA solution was then neutralized with 160 l of 0.1 M HEPES, pH 7.4.

[0222] Immobilization of oligonucleotide probes (primers, SEQ ID NO:8)was performed as follows. A total of 100 l of 3× PBS buffer containingthe primers (150 nM) was dropped into each well of a 96-well microtiterplate (Fisher Sci., Suwanee, Ga.). After incubation for 2 h at 37° C. orovernight at room temperature, the plate was washed three times with 1×PBS buffer containing 0.05% (w/vol) Tween 20. The oligonucleotide-coatedplates were stable for 2 months at 4° C.

[0223] Direct PCR reactions were performed in a final volume of 50 l(Sisk R B. in book: Molecular diagnostics: for the clinicallaboratorian. Ed. by Coleman W B., and Tsongalis G J. Humana Press Inc.,Totowa, N.J. 1997, pp.103-121). The total DNA (5 l), isolated from bloodsamples of individuals, to oligonucleotide-coated plate in dublicatesand 45 l of master ready-to-use buffer containing 1 l TaKaRa Z-Taq DNApolymerase (TaKaRa Biomedicals, Otsu, Shiga, Japan) 10 l AMV/Tfl 5×reaction buffer, 1 l dNTP mix (Promega, Madison, Wis.) 2 l of 25 mMMgSO₄ were added and sealed. The 30-thermal cycles (98° C.—5 s, 66° C.—2sec) amplification using programmable Gene Cycler thermocycler (Bio-RadLab., Hempstead, UK) for 20 minutes was performed. Then 50 l ofPicoGreen reagent (Mol.Probes, Inc., Eugene, Oreg.) were added to eachPCR products and mixed on a shaker (BioTechniques 20:676 (1996). Sampleswere incubated 5 min at room temperature, protected from light. Afterincubation the fluorescence of the samples was measured using afluorescence microplate reader (Mol.Device, Sunnyvale, Calif.) andstandard fluorescein wavelengths (excitation 480 nm, emission 520 nm).The fluorescence value of the reagent blank was subtracted from that ofeach of the samples, and the data was employed to generate five-pointstandardization curves of fluorescence versus DNA concentration, from 25pg/ml to 25 ng/ml reaction of control target cDNA (50 ng/ml stock) withthe same Pico Green reagent.

[0224] The other method of total DNA isolation is follows. Whole bloodwas spotted onto FTA paper and lysed, and samples of DNA immobilizedwithin the matrix of the stain card were punched into a 3 mm (⅛″)diameter paper (1 mm or 2 mm Harris Micro-Punch™) and amplified directlyby the amplification mix (Mackey K. et al. Mol.Biotechnol. 9: 1-5(1997).

[0225] The FTA Bloodstain Card is divided into 4 circles for at least 4different 120 l samples of EDTA collected whole blood. Samples of bloodwere dried at room temperature for at least 1 hour. A circle was drawnwith a #2 pencil around each blood to visualize where the blood had beenspotted after the FTA paper processing. The FTA Bloodstain Card was thenplaced in a small plastic tray and 50 ml of FTA Purification Reagent wasadded and incubated on a shaker for 5 minutes. FTA Purification Reagentwas replace 3 times with 25-50 ml of the fresh solution and shaken foran additional 5 minutes. Then 25-50 ml of TE-4 (10 mM Tris-HCl pH 8.0;0.1 mM EDTA pH 8.0) was added and the mixture incubated twice on ashaker for 5 minutes. The FTA Bloodstain Card was allowed to air drycompletely during 2 hours at room temperature. The samples were thenpunched from the cards using a 3 mm diameter punch or the HarrisMicro-Punch (1.2 mm or 2.0 mm), and transferred into correspondingmicroplate wells. PCR was then performed using the above-describedprocedure using regular PCR microplates and a ready-to-use buffercontaining primers.

[0226] Patients (n-30, the age of 44-77) were divided into two groups.The first group of patients (n=12) were diagnosed with TIA in thecarotid circulatory system, according to the following neurologicalcriteria. Neural dysfunction was localized to a specific vasculardistribution; the duration of the attack was usually less that 15minutes and never exceeded 24 hours; and the patients did not haveabnormal neurologic signs between attacks. The second pre-stroke group(n=18) were diagnosed with TIA in the vertebral-basilar circulatorysystem. The second group of patients was subdivided on the basis ofcompensation or non-compensation of neurological deficit. The thirdgroup (n=12) included patients with migraine and epilepsy.

[0227] The control group of healthy individuals (n=20) showed a level ofNR2A cDNA expression of 1.2 0.11 pg/ml. The first group demonstratedslightly elevated levels of NR2A cDNA expression of 1.7 0.13 pg/ml. Thepatients with compensation of neurological deficit from the second groupshowed a level of NR2A cDNA expression of 1.8 1.4 pg/ml. At the sametime, the patients without compensation of neurological deficit thatpossessed more severe symptoms of TIA showed levels of NR2A cDNAexpression of 3 times the levels seen in healthy individuals. Patientssuffering migraine and epilepsy did not show any increase of NR2A cDNAexpression when compared with the control group.

Example 10 Immunological Analysis of Blood Serum Specimens

[0228] Blood samples (10 ml, collected by venipuncture) from patientswith cerebral ischemia (n=70), and healthy individuals (n=200),collected according to standard clinical protocols, were examined at theNeurology Hospital of Russian Medical Academy (Moscow, Russia). Theblood specimens were centrifugated (4000 g, 5 min, +4° C.) and thecollected serum stored at −70° C. for further analysis.

[0229] Computer analysis was employed to predict the antigenicdeterminants in the NR2A receptor protein structure based onhydrophobicity profile (Hopp, T. P. and K. R. Woods, Proc. Natl. Acad.Sci. USA 6:3824-3828 (1981)) and antigenicity (Welling, G. W., et al.,FEBS Lett. 188:215-218 (1985)). Based upon this analysis, the N-terminalsequence of the NR2A NMDA receptor was synthesized. This syntheticpeptide, which corresponded to amino acid sequence (494-514) (Grandy, DK., et al, Proc. Natl. Acad. Sci. U.S.A. 86:9762-9766(1989) (SEQ IDNO:3) of human NR2A NMDA, was produced by solid-phase synthesis in aNPS-400 semi-automated synthesizer (Neosystem Lab, France) on MBHA resinusing the BOC/Bzl strategy for the first two amino acids. The peptideswere purified by preparative HPLC on a DELTAPAC™C18 column (WatersChromatography, Milford, Mass.) in a H₂O/acetonitrile/0.015 TFA system.The purity of the peptides was determined by analytical HPLC and rangedfrom 90% to 98%. The peptide sequence was verified by amino acidanalysis after acid hydrolysis. This peptide was used in immunoassays ofblood serum from patients and healthy individuals.

[0230] A quantitative analysis of the level of NR2A autoantibodies inserum samples was performed by enzyme-linked immunosorbent assay (ELISA)(Ngo, T. T. and H. M. Lenhoff, FEBS Lett. 116:285-288 (1980)). Thediluted blood sera (1:50) and polyclonal antibodies to the NR2A peptideas a standard (0.01 ng/ml-400 ng/ml) were applied to the immunosorbent.The plate was incubated for 1 h at 25° C. and then washed by 0.05 Mphosphate buffer, pH 7.4, containing 0.05% of TWEEN-20™. Rabbitantibodies to the human immunoglobulin labeled with horseradishperoxidase were added (Sigma, St. Louis, Mo.; 1:1000), and the plate wasincubated for 1 h at 25° C. After incubation the wells were washed twicein the same buffer. The reaction was revealed by o-phenylenediamine in0.05 M citrate buffer, pH 4.3 monitored at 490 nm on a microplate reader(BioRad, UK). The titer of NR2A autoantibodies in blood serum wasdetermined by ELISA using a standard curve of the absorbence units ofNR2A autoantibodies versus their concentration in a microtiter wellplate.

[0231] The synthetic peptide corresponding to the NR2A NMDA glutamatereceptors (3 μg) were immobilized on a nitrocellulose membrane (0.45 μm,Shleicher-Shuell, Germany) in phosphate-buffered saline (PBS), pH 7.4,then washed 2-3 times in the same buffer. Membranes with immobilizedpeptide were incubated with the diluted serum (1:50) of cerebralischemia patients and other subjects for 1 h at 25° C., and then rinsed4 times with the PBS buffer. Secondary rabbit anti-human immunoglobulinsconjugated with horseradish peroxidase (Sigma, St. Louis, Mo.; 1:1000)were incubated with the membrane for 1 h at 25° C., then washed 4 timeswith PBS. The development of brown color was registered and thenquantitated by densitometry.

[0232] To provide a positive control or standard, rabbit polyclonalantibodies were raised against NR2A synthetic peptide corresponding toamino acid sequence predicted from the cloned human NR2A protein(Science 256:1217-1221 (1992); SEQ ID NO:1). For glutaraldehydeconjugation, 10 mg of peptide and 40 mg of human serum albumin (Sigma,St. Louis, Mo.) were incubated for 1.45 h at room temperature in 4 ml ofPBS containing 5% glutaraldehyde. The reaction was stopped by addingglycine to a final concentration of 0.2 M, and the conjugate wasdialyzed against PBS. Rabbits were given initial injections of 1 mg ofconjugated peptide in complete Freund's and subsequent injections of 0.5mg of peptide in incomplete Freund's adjuvant at successive 2 weekintervals. Antibodies were affinity purified according standardprocedure (Warr, G. W., Purification of antibodies, In: Antibody as aTool, Eds., Marchalonis, J. J., and G. W. Warr, J. Wiley, UK, pp. 59-96(1982)) and were shown to be selective for the NR2A NMDA glutamatereceptor using Western blot analysis.

[0233] The patients (men, n=30; women, n=40; age of 40-75) were admittedin the hospital within no more than six hours after the onset of anishemic episode. All patients were divided into three groups accordingto the severity of the stroke: The first group had moderate ischemicstroke (n=25), manifested by moderate focal deficit (>60—Orgogozoscale). The second group had severe stroke (n=30), manifested by milddisorders of consciousness, severe headache, meningeal sings, andpronounced focal deficit (30-60—Orgogozo scale). The third group hadextremely severe stroke (n=15), accompanied by stupor-coma, signs ofbrain edema, autonomic dysfunction, and severe focal deficit(<30—Orgogozo scale).

[0234] The level of NR2A autoantibodies was measured in the blood serumof healthy persons (n=200, age 35-75) as a control, and ranged from0.3-1.5 ng/ml. The NR2A autoantibody level in the 55 patients of thefirst and second groups was significantly greater than that in thecontrol group (p<0.01). Levels of NR2A autoantibodies were monitoredevery three hours during the first day, and then up to 5th day afterstroke. The level of NR2A autoantibodies in the blood serum of patientswith severe stroke was significantly higher than that in the blood serumof patients with moderate stroke, especially in the 9-12 hours after theonset of a stroke (p<0,05). The tendency for NR2A autoantibodies levelto decrease to the control level on the first day of stroke wasregistered in group of patients with good neurological recovery (90,50,5units on Orgogozo scale). It can be concluded that the dynamic changesin NR2A autoantibodies level may predict a recovery period of patientsafter ischemic stroke.

Example 11 SPRIA Assay of Autoantibodies

[0235] The solid-phase radioimmunoassay (SPRIA) of autoantibodies isperformed as follows: a 10% acetic acid solution is added for one minuteto the Cooker microtiter microplates (available from Dynatech Co., USA)for activation, whereupon 0.1 ml of the blood serum under analysis(diluted 1:40) is applied to the microplates and subjected to incubationfor four hours at 25° C. Then the microplate are washed with a 0.14 Msodium chloride solution and 0.1 ml of a mixture of the respectivefragment of the mammal's brain protein labeled by 125I in the presenceof nonlabelled one. The plates are incubated for 20 hours at 4° C. Oncompletion of incubation, the microplates are washed with a 0.14M sodiumchloride solution, after which each of the wells of the microplates iscut off and placed in gamma-counting vials.

Example 12 ELISA Assay of Autoantibodies

[0236] The enzyme-linked immunosorbent assay (ELISA) of autoantibodiesis carried out as follows: the samples of the blood serum diluted 1:40or 1:50 are applied to the respective immunosorbent. Then the platecarrying the immunosorbent is incubated for 30 min at 37° C., whereuponthe wells of the plate are washed with a 0.05 M phosphate buffer,containing 0.05% of Tween-20. Rabbit antibodies to human immunoglobulinlabeled with horseradish peroxidase (conjugate) are added thereto, andthe plate is reincubated for 35 min at 37° C., then washed by theaforementioned buffer and distilled water. The reaction with conjugateis determined by adding chromogen, i.e., orthophenylenediamine in thepresence of 30% hydrogen peroxide. The intensity of color development isevaluated by using the rider (available Multiskan microplate rider) atthe 492 nm wavelength.

[0237] It will be apparent to those skilled in the art that variousmodifications and variations can be made in the present inventionwithout departing from the scope or spirit of the invention. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

What is claimed is: 1) A method for diagnosing a central nervous systemdisorder comprising measuring the level of NR2A and/or NR2B NMDAreceptor or fragment thereof in a biological sample from a humansubject, other than by measuring autoantibodies against a full sequenceNR2A and/or NR2B receptor. 2) The method of claim 1 wherein the centralnervous system disorder is TIA or stroke or the risk of TIA or stroke.3) The method of claim 1 further comprising comparing the level of NR2Aand/or NR2B NMDA receptor or fragment thereof in the biological sampleto a baseline level selected from population norms and prior levelsmeasured for the subject. 4) The method of claim 1 wherein the the levelof NR2A and/or NR2B NMDA receptor or fragment thereof is measureddirectly from the level of the N-terminal domain of a NR2A and/or NR2BNMDA receptor or fragment thereof in the biological sample. 5) Themethod of claim 1 wherein the level of NR2A and/or NR2B NMDA receptor orfragment thereof is measured indirectly from the level of antibodiesagainst the N-terminal domain of a NR2A and/or NR2B NMDA receptor,analogue thereof, or fragment thereof in the biological sample. 6) Themethod of claim 1 wherein the level of NR2A and/or NR2B NMDA receptor orfragment thereof is measured indirectly from the mRNA encoding the NR2Aand/or NR2B NMDA receptor or fragment thereof in the biological sample.7) The method of claim 1 wherein the the level of NR2A and/or NR2B NMDAreceptor or fragment thereof is measured directly from the level of thepeptide represented by SEQ ID NO:2, 3, 11, and/or
 12. 8) The method ofclaim 1 wherein the the level of NR2A and/or NR2B NMDA receptor orfragment thereof is measured indirectly from the level of antibodiesagainst a peptide of SEQ ID NO:2, 3, 11, and/or 12, analogue thereof, orfragment thereof, in the biological sample. 9) The method of claim 1wherein the the level of NR2A and/or NR2B NMDA receptor or fragmentthereof is measured indirectly from the level of cDNA corresponding toSEQ ID NOS: 5, 6, 7, 14, and/or 15, analogue thereof, or fragmentthereof, in the biological sample. 10) The method of claim 1 fordiagnosing the existence of TIA or stroke, further comprisingwithdrawing the biological sample from a human subject, wherein thebiological sample is withdrawn within three hours of the onset ofsymptoms of TIA or stroke. 11) The method of claim 10 for diagnosing theexistence of TIA or stroke, wherein the amount of time elapsed betweenwithdrawing the biological sample from the subject, and detecting ormeasuring the presence or quantity of the NR2A and/or NR2B NMDA receptoror fragment thereof, is less than about one hour. 12) The method ofclaim 1 for diagnosing the existence of TIA or stroke furthercomprising, when the diagnosis confirms a stroke, evaluating from thelevel of NR2A and/or NR2B NMDA receptor or fragment thereof whether thestroke is ischemic or hemmorhagic and administering ischemic orhemmorhagic stroke therapy as appropriate. 13) The method of claim 1 fordiagnosing the existence of TIA or stroke further comprising, if TIAand/or stroke is confirmed, evaluating from the level of NR2A and/orNR2B NMDA receptor or fragment thereof whether the episode is TIA orstroke and administering TIA or stroke therapy as appropriate. 14) Themethod of claim 1 for diagnosing the existence of TIA or stroke furthercomprising, if TIA and/or stroke is confirmed, evaluating from the levelof NR2A and/or NR2B NMDA receptor or fragment thereof cranial infarctvolume, and administering therapy consistent with the extent of cranialinfarct. 15) The method of claim 1 wherein the NR2A and/or NR2B NMDAreceptor or fragment thereof is measured by immunoassay. 16) The methodof claim 1 wherein the NR2A and/or NR2B NMDA receptor or fragmentthereof is detected or measured by agglutination comprising: a)contacting the biological sample with poly- or monoclonal antibodiesbound on an agglutinating carrier for sufficient time and underconditions to promote agglutination, wherein the antibodies are specificfor the NR2A and/or NR2B NMDA receptor, analogue thereof, or fragmentthereof; and b) reading a signal generated from the agglutination;wherein the signal correlates to the titer of NR2A and/or NR2B NMDAreceptor or fragment thereof present in the sample. 17) The method ofclaim 16, wherein the sufficient time period is 30 minutes or less. 18)The method of claim 16 wherein the carrier comprises polysterene latexbeads having a mean diameter of from about 0.25 to about 0.4 μm. 19) Themethod of claim 1, wherein the biological sample is blood, urine, bloodplasma, blood serum, cerebrospinal fluid, saliva, perspiration or braintissue, or a derivative thereof. 20) The method of claim 1, wherein thebiological sample is blood diluted to a ratio of from about 1:2 to about1:32. 21) The method of claim 1, wherein levels of cDNA are measured by:a) complexing the biological sample with one or more oligonucleotideprimers to the cDNA for a sufficient time period and under conditions topromote amplification; b) contacting the complex with an indicatorreagent comprising a secondary oligonucleotide complementary to the cDNAattached to a signal-generating compound; and c) measuring the signal.22) The method of claim 21, wherein the signal-generating compound isselected from the group consisting of horseradish peroxidase, alkalinephosphatase, urinase and a non-enzymatic reagent. 23) The method ofclaim 21, wherein the cDNA comprises a poly- or oligonucleotide of SEQID NO:6 or 14, or a fragment thereof. 24) The method of claim 21,wherein the cDNA comprises a poly- or oligonucleotide of SEQ ID NO:7 or15, or a fragment thereof. 25) The method of claim 21, wherein the oneor more oligonucleotide primers are of SEQ ID NO:8, 9, 16, and/or 17.26) The method of claim 1 wherein levels of NR2A and/or NR2Bautoantibody are measured comprising: a) contacting the biologicalsample with a protein fragment of the N-terminal domain of the NR2Aand/or NR2B receptor for a time sufficient and under conditions to forma complx between autoantibodies in the sample and the protein fragment;b) contacting the complex with an indicator reagent comprising asecondary antibody attached to a signal generating compound; and c)measuring the signal. 27) The method of claim 26, wherein the secondaryantibody is specific for the protein fragment. 28) The method of claim26, wherein the secondary antibody is specific for the autoantibodybeing measured. 29) The method of claim 1, further comprising measuringone or more other biomarkers for TIA or stroke in the biological sample.30) The method of claim 29 wherein the one or more biomarkers comprisesan agonist or antagonist of an NMDA receptor. 31) The method of claim 29wherein the one or more other biomarkers comprises glutamate orpolyglutamate. 32) The method of claim 29 wherein the one or more otherbiomarkers comprises a thromboembolic biomarker. 33) The method of claim29 wherein the one or more other biomarkers comprises homocysteine orpolyhomocysteine and glutamate or polyglutamate. 34) The method of claim1 further comprising, if the biological sample comprises a titer higherthan 3.34 for glutamate, 2.23 for homocysteine and 2.63 for NR2A-B,administering TIA/stroke therapy or stroke risk reduction therapy. 35)The method of claim 1, further comprising, if the levels of NR2A cDNA inthe subject are greater than about 1.0 pg/ml, administering TIA/stroketherapy or stroke risk reduction therapy. 36) The method of claim 1,further comprising, if the levels of NR2Aantibodies exceed 1.0 ng/ml,administering TIA/stroke therapy or stroke risk reduction therapy. 37)The method of claim 1 for diagnosing the risk of suffering TIA orstroke, in a subject not then exhibiting symptoms of TIA or stroke. 38)The method of claim 1 for diagnosing the progression of TIA or strokefurther comprising measuring the level of NR2A and/or NR2B NMDA receptoror fragment thereof in a biological sample one or more additional times,at a frequency of less than about 6 hours. 39) The method of claim 38wherein TIA or stroke therapy is concurrently being administered to thesubject. 40) The method of claim 1 for diagnosing the remission of riskfor stroke further comprising measuring the level of NR2A and/or NR2BNMDA receptor or fragment thereof in a biological sample one or moreadditional times, in a subject to whom stroke risk management therapy isadministered. 41) A method for diagnosing a central nervous systemdisorder comprising directly or indirectly measuring: a) the level ofNR2A or NR2B NMDA receptor or fragment thereof in a subject; and b) thelevel of one or more agonists or antagonists of the NR2A and/or NR2Breceptor. 42) The method of claim 41 wherein the one or more agonists orantagonists comprise glutamate, polyglutamate, homocysteine and/orpolyhomocysteine. 43) The method of claim 41 wherein the level of NR2Aor NR2B NMDA receptor or fragment thereof in the subject is measureddirectly from the amount of NR2A or NR2B NMDA receptor or fragmentthereof present in the biological sample. 44) The method of claim 41wherein the level of NR2A or NR2B NMDA receptor or fragment thereof in asubject is measured indirectly from the amount of NR2A or NR2B NMDAreceptor mRNA present in the biological sample. 45) The method of claim41 wherein the level of NR2A or NR2B NMDA receptor or fragment thereofin the subject is measured from the amount of autobody against NR2A orNR2B NMDA receptor or fragment thereof in the biological sample. 46) Acomposition comprising a fragment of a polynucleic acid encoding theNR2A or NR2B subunit, wherein the fragment encodes the N-terminal domainof the NR2A or NR2B NMDA receptor. 47) A composition comprising anoligo- or polynucleotide selected from the group consisting of SEQ IDNO:6, 7, 8, 9, 15, 16, and 17, or a fragment thereof. 48) A proteinfragment comprising the N-terminal domain of the NR2A or NR2B NMDAreceptor. 49) A peptide or polypeptide of sequence listing SEQ ID NO: 2,3, 4, 11, 12, or
 13. 50) A test kit for detecting NR2A and/or NR2B cDNAamplification comprising: a) NR2A and/or NR2B cDNA primer attached to asolid phase; and b) an indicator reagent comprising a secondaryoligonucleotide complementary to NR2A and/or NR2B cDNA, bound to asignal-generating compound capable of generating a measurable signal.51) The test kit of claim 50, wherein the solid phase is a polymermatrix. 52) The test kit of claim 50, wherein the signal-generatingcompound is selected from the group consisting of horseradishperoxidase, alkaline phosphatase, urinase and non-enzymatic reagents.53) A kit comprising: a) poly- or monoclonal antibodies to NR2A and/orNR2B proteins immobilized on a carrier; and b) a control solution. 54)The kit of claim 53 further comprising poly- or monoclonal antibodies toa second biomarker for a central nervous system disorder immobilized ona carrier. 55) The kit of claim 53 wherein the second biomarkercomprises glutamate, polyglutamate, homocysteine, or polyhomocysteine.56) The kit of claim 53 further comprising poly- or monoclonalantibodies to second and third biomarkers for a central nervous systemdisorder immobilized on carriers, wherein the second biomarker comprisesglutamate or polyglutamate, and the third biomarker compriseshomocysteine or polyhomocysteine. 57) A kit comprising: a) proteinfragments comprising the N-terminal domain of a NR2A and/or NR2B NMDAreceptorpoly- or monoclonal antibodies immobilized on a carrier; and b)an indicator reagent comprising a secondary antibody attached to asignal generating compound. 58) The kit of claim 57, wherein thesecondary antibody is specific for the protein fragment. 59) The kit ofclaim 57, wherein the secondary antibody is specific for theautoantibody being measured. 60) A composition comprising a poly- ormonoclonal antibodies against the NR2A or NR2B NMDA receptor or fragmentthereof, covalently bound to latex beads. 61) The composition of claim60 wherein the poly- or monoclonal antibodies are raised against theN-terminal domain of an NR2A or NR2B NMDA receptor or fragment thereof62) The composition of claim 60 wherein the latex beads are polystyrenelatex beads having a mean diameter of from about 0.25 to about 0.4 μm.